E. coli separatome-based protein expression and purification platform

ABSTRACT

Provided is a separatome-based peptide, polypeptide, and protein expression and purification platform based on the juxtaposition of the binding properties of host cell genomic peptides, polypeptides, and proteins with the characteristics and location of the corresponding genes on the host cell chromosome of  E. coli . The separatome-based protein expression and purification platform quantitatively describes and identifies priority deletions, modifications, or inhibitions of certain gene products to increase chromatographic separation efficiency, defined as an increase in column capacity, column selectivity, or both, with emphasis on the former. Moreover, the separatome-based protein expression and purification platform provides a computerized knowledge tool that, given separatome data, and a target recombinant peptide, polypeptide, or protein, intuitively suggests strategies facilitating efficient product purification. The separatome-based protein expression and purification platform is an efficient bioseparation system that intertwines host cell expression systems and chromatography.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. provisionalapplication Ser. No. 61/878,882, filed Sep. 17, 2013, the contents ofwhich are herein incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT ANDJOINT RESEARCH AGREEMENT DISQUALIFICATION UNDER THE CREATE ACT(COOPERATIVE RESEARCH AND TECHNOLOGY ENHANCEMENT ACT OF 2004 (CREATEACT) (PUB. L. 108-453, 118 STAT. 3596 (2004))

This invention was made with government support under grants Nos.0534836, 0533949, 1237252, 1142101, and 1048911, awarded by the NationalScience Foundation. The U.S. government has certain rights in theinvention.

The present invention was collaboratively made by scientists from theUniversity of Arkansas and the University of Pittsburgh under theabove-noted joint NSF grants that were in effect on or before the datethe presently claimed invention was made. The claimed invention was madeas a result of activities undertaken within the scope of the jointresearch agreement. The term “joint research agreement” means the jointNSF research grants awarded to the above-noted parties for theperformance of experimental, developmental, or research work in thefield of the claimed invention.

BACKGROUND

Disclosed herein is a proteomics-based protein expression andpurification platform, more particularly a single cell line, or set ofcell lines, designed by manipulating the separatomes associated withvarious separation techniques, in particular column chromatography, thatcan be used in a wide variety of processes for the expression ofrecombinantly produced peptides, polypeptides, and proteins, and to thesubsequent rapid, efficient, and economical recovery thereof in highyield, thereby eliminating the need to develop individualized host cellsfor each purification process.

Current society is heavily dependent on mass-manufactured peptides,polypeptides and proteins that are used in everything from cancertreatment medications to laundry detergents. More than 325 millionpeople worldwide have been helped by the over 155 recombinantly producedpolypeptides and peptides (drugs and vaccines) currently approved by theUnited States Food and Drug Administration. In addition, there are morethan 370 biotechnology drug products and vaccines (“biologics”)currently in clinical trials targeting more than 200 diseases, includingvarious cancers, Alzheimer's disease, heart disease, diabetes, multiplesclerosis, immunodeficiency, and arthritis. Enzymes used in industrialprocesses claim approximately a $2.7 billion dollar market, with anexpected growth to a value of S6 billion dollars by 2016. Of theapproximately 3000 industrial enzymes in use today for applications inbiotechnology, food, fuel, and pulp and paper industries, aboutone-third of these are produced in recombinant bacteria.

Manufacturing of therapeutically useful peptides, polypeptides, andproteins has been hampered, in large part, by the limitations of theorganisms currently used to express these molecules, and by the oftenextensive recovery steps necessary as the final product is isolated.Recombinant protein expression is the preferred, predominant method forthe manufacture of these pharmaceuticals, herein referred to as“biologics” to differentiate them, in particular, both from chemicallysynthesized therapeutics (e.g., antihistamines or CNS drugs) and fromindustrial enzymes such as pectinases or restriction endonucleases, forexample. In general, the purification of a biologic to within tolerablelimits is the most costly stage of manufacturing and validation, withthe burden of regulation placed upon it by the Food and DrugAdministration (FDA) or similar (inter)national entities. RecombinantDNA techniques, hybridoma technologies, mammalian cell culturing,metabolic engineering, and fermentation improvements have permittedlarge-scale production of biologics.

As large-scale production issues are solved, manufacturing steps thatlimit productivity are shifted downstream. In an effort to quickentime-to-clinic and market, research efforts have focused on cuttingmaterial costs, improving productivity at large-scale, and developingrobust, generic separation steps. In the biologics manufacturingprocess, cell lines are cultivated to produce, or express, the biologic;during this process, the desired biologic is expressed alongsideunwanted host cell proteins. These contaminants then have to beseparated from the biologic through expensive and time-consumingmulti-step purification processes that often include centrifugation,ultrafiltration, extraction, precipitation, and the cornerstone ofbioseparation, chromatographic separation. Since downstream processesaccount for 50% to 80% of total manufacturing costs, efforts to optimizepurification of high-value, high-quality products are critical tosuccess in the biopharmaceutical industry. For example, if there is amodest 5% loss of biologic per purification step, final yields of about70% are encountered should the processing require 5 to 8 downstreamsteps. This overall loss is intolerable as market demands for biologicsincrease. End-uses for peptides, polypeptides, and proteins producedrecombinantly, other than biologics, include, but are not limited to,diagnostic kits (e.g., glucose dehydrogenase for glucose sensing),enabling technologies (e.g., ligases for recombinant DNA efforts),consumer products (e.g., proteases for laundry soap), manufacturing(e.g., isomerases for production of corn syrup), and biofuel generation(e.g., cellulases for switchgrass processing). Materials of theseproduct categories also suffer from the desire for efficient downstreamprocessing, although their product validation is less stringent than fora biologic.

For the illustrations above, both recovery from the culture andpurification are paramount. Challenges to the industry standardtechnique of column chromatography, a critical element to mostbioseparation schemes, are dictated by lack of separation efficiency,the variety of chromatography separation media, and the diversecomposition of the mobile phase. Lack of separation efficiency manifestsitself predominantly as a reduction in column capacity, defined as theamount of target molecule bound per adsorption cycle, and selectivity,defined as the amount of target molecule bound divided by the totalamount of material bound per adsorption cycle. The traditional method ofaddressing separation efficiency is empirical, and is driven by pastexperience because no software design tool, similar to CHEMCAD (chemicalengineering) and SPICE (electrical engineering), for bioseparationprocess design exists in the public domain, if at all. Therefore, anyimprovements in the recovery of peptides, polypeptides, or proteins interms of an increase in separation efficiency, column capacity inparticular, have been traditionally gained by improvements in theproperties of the chromatographic adsorbent, by artful design of thegradient used to elicit separation, or in some cases, by the enhancementof binding through the addition of His₆, maltose binding protein, Arg₈,or similarly designed affinity tails or tags. Although affinity tails ortags are widely used for purification of recombinant proteins, inparticular through the use of His₆, the continued presence of genomicpeptides, polypeptides, and proteins exhibiting affinity for the resinsused in these chromatographic methods remains problematic. Notably, whenhost cell genomic peptides, polypeptides, and proteins are retained inthe adsorption step, significant losses in column capacity andcomplications in gradient elution occur. Selection of companionchromatographic steps in a rational manner to increase separationefficiency, i.e., separation capacity (product recovery), separationselectivity (product purity), or both, is nearly impossible due to lackof knowledge regarding the contaminant species, and is thereforedeveloped somewhat arbitrarily, requiring tedious, time-consuming, andexpensive trial and error experimentation.

As disclosed herein, one route to supplement traditional means to aid inthe purification of peptides, polypeptides, or proteins would be toalter the proteome of the host cell in order to reduce the burden ofhost cell contaminant adsorption. This concept is orthogonal to theseries of patents and applications by Blattner et al. that disclose anumber of different strains of E. coli engineered to contain reducedgenomes—in contrast to the proteome—to facilitate the production ofrecombinant proteins (U.S. Pat. Nos. 8,178,339; 8,119,365; 8,043,842;8,039,243; 7,303,906; 6,989,265; US20120219994A1; and EP1483367B1). U.S.Pat. No. 8,119,365 claims E. coli wherein the genome is between 4.41 Mband 2.78 Mb. U.S. Pat. No. 8,043,842 claims E. coli wherein the genomeis between 4.27 Mb and 4.00 Mb. U.S. Pat. No. 8,039,243 claims variouslybetween 4.41 and 3.71 Mb, 4.31 Mb and 3.71 Mb, and 4.27 Mb and 3.71 Mb.U.S. Pat. No. 6,989,265 discloses E. coli wherein the genome is at least5% to at least 14% smaller than the genome of its native parent strain.EP1483367B1 claims E. coli having a chromosome that is geneticallyengineered to be 5% to 40% smaller than the chromosome of its nativeparent E. coli strain.

These documents variously discuss the concepts of reduced genome E. colifor use in the production of recombinant proteins, improving recombinantprotein expression in E. coli by improving the growth/yield propertiesand robustness as a recombinant host by eliminating large numbers ofnon-essential genes and improving E. coli transformation competence.Expression of endogenous/native proteins in host cells is also presumedto be reduced. None of these documents either discloses or discusseschromatographic purification procedures, or the optimization thereof inconjunction with the design of optimized host cells, to improveseparation efficiency leading to a purified or partially purified targetpeptide, polypeptide, or protein.

U.S. 2009/0075352 discloses the use of in silico comparative metabolicand genetic engineering analyses to improve the production of usefulsubstances in host strains by comparing the genomic information of atarget strain for producing a useful substance to the genomicinformation of a strain that overproduces the useful substance byscreening for, and by deleting genes unnecessary for the overproductionof the useful substance, thereby improving product yield. This workillustrates metabolic engineering efforts directed to small moleculeproduction (succinic acid), and as in the case of the patent documentsdiscussed above, this application does not disclose or discusschromatographic purification procedures, or the optimization thereof toimprove separation efficiency leading to a target peptide, polypeptide,or protein.

Yu et al. (2002) Nature Biotechnol. 20:1018-1023 discloses a method fordetermining essential genes in E. coli and minimizing the bacterialgenome by deleting large genomic fragments, thereby deleting genes thatare nonessential under a given set of growth conditions and identifyinga minimized set of essential E. coli genes and DNA sequences. Neitherthe term “chromatography” nor “purification” is mentioned.

U.S. application 2012/0183995 discloses genetic modification of Bacillusspecies to improve the capacity to produce expressed proteins ofinterest, wherein one or more chromosomal genes are inactivated ordeleted, or wherein one or more indigenous chromosomal regions aredeleted from a corresponding wild-type Bacillus host chromosome. Thisincludes removing large regions of chromosomal DNA in a Bacillus hoststrain wherein the deleted indigenous chromosomal region is notnecessary for strain viability. These modifications enhance the abilityof an altered Bacillus strain to express a higher level of a protein ofinterest over a corresponding non-altered Bacillus host strain. Thisapplication does not discuss improved chromatographic separation ofexpressed target recombinant peptides, polypeptides, or proteins fromendogenous Bacillus proteins.

Asenjo et al. (2004). “Is there a rational method to purify proteins?From expert systems to proteomics”, Journal of Molecular Recognition17:236-247, discusses optimizing protein purification steps based onknowledge of the physicochemical properties of the target proteinproduct and the protein contaminants. The paper notes “the rule of thumbthat reflects the logic of first separating impurities present in higherconcentrations.” The concept of reduced genome host cells is notdisclosed.

While the above-mentioned patents and journal articles do not discloseor discuss chromatographic purification procedures or the improvement ofchromatographic separation efficiency, other references either outlinethe general process by which data on host cell proteins that interactwith chromatography media can be obtained, or focus on the eliminationof product-specific impurities through gene knockout. Cai et al. (2004)Biotechnol. Bioeng. 88:77 and Tiwari et al. (2010) Protein Expressionand Purification 70:191-195 disclose the application of cellularextracts of E. coli to various affinity and non-affinity chromatographicmedia, and the identification of adsorbed proteins by mass spectroscopyand 2D gel electrophoresis. While the metabolic characteristics of theproteins encountered were discussed, these references do not discloseany indications of improvement in separation efficiency. Liu et al.(2009) J. Chromatog. A 1216:2433-2438, Bartlow et al. (2011) ProteinExpression and Purification 78:216-224, and Bartlow et al. (2012)American Institute of Chemical Engineers Biotechnol. Prog. 28:137-145disclose the potential for improvement in product quality, purity inparticular, should genes that express proteins that co-elute with aspecific protein, i.e., histidine-extended Green Fluorescent Protein, bedeleted from the chromosome of E. coli. The quantitative data in thisseries of papers do not disclose or suggest improvements that lead to anincrease in column capacity, nor do they demonstrate or suggestimprovements that point to a universally applicable host strain withimproved properties, useful for producing a variety of differentpeptides, polypeptides, or proteins, be they extended with an affinitytail or tag (or not). Indeed, should the genes identified and deemedimportant in Liu et al. (2009), supra, be deleted, an increase ofsignificantly less than one percent (1%) in column capacity would beachieved. A similar argument for the deletion of genes responsible forproduct-specific contaminants applies to Caparon et al. (2010)Biotechnol. Bioeng. 105(2):239-249. This article discloses four specificgene deletions that improve the purity of the final biologic, sincethree of the proteins co-elute with the target and a fourth causesproteolytic degradation of the biologic. Lacking in this reference is ameans of applying quantitative metrics to prioritize efforts that leadto increases in separation efficiency independent of target peptides,polypeptides, and proteins, and a method to interpret these data toprepare a host cell or set of host cells that provide increases inseparation efficiency for as many different target molecules aspossible.

In view of the foregoing, there exists a need for improved methods forrecovering in quantity, and purifying, recombinant target peptides,polypeptides, and proteins from E. coli and other host cells routinelyused for recombinant expression of, for example, therapeuticproteinaceous molecules and industrial enzymes. Development ofbioseparation regimens can be challenging, requiring somewhat arbitrarytrial and error combination of conventional chromatographic methods. Thepresence of host cell peptides, polypeptides, and proteins reducesseparation step efficiency (adsorption and elution), and the tradeoffbetween overall yield and purity may not be optimal. Alternately,although the use of an affinity tail helps reduce the chromatographicspace explored, it can still be plagued by co-adsorbing/co-elutingmolecules, requiring further purification steps; addition/removal of theaffinity tail via digestion steps; and cost (ligand and endonuclease).

The methods and host cells disclosed herein address these problems andmeet these needs. These methods and host cells provide a novel route tosupplement or supplant conventional methods to aid in the purificationof target recombinant peptides, polypeptides, and proteins. This isaccomplished by providing a rational scheme for altering the proteome ofhost cells used for expression in order to reduce the burden ofadsorption of host cell peptides, polypeptides, and proteins that mayinterfere with the recovery and purification of any target molecule.This is accomplished by first identifying the separatome, defined as asub-proteome associated with a separation technique, columnchromatography for example, by applying a formal method thatmathematically prioritizes specific modifications to the proteome via,for example, gene knockout, gene silencing, gene modification, or geneinhibition, and designing host cells with the desired property ofimproved chromatographic separation based on this information. Hostcells, or sets of host cells, as disclosed herein display a reducedseparatome, the properties of which lead to an increase in columncapacity as peptides, polypeptides, or proteins with high affinity areeliminated first. Uniquely focusing on host cell peptides, polypeptides,or proteins with high affinity, rather than those with affinity similarto, or less than a presumed target recombinant molecule, facilitates aset of modifications that are useful for improving separation efficiencyfor a wide range of peptides, polypeptides, or proteins. Such highaffinity host cell peptides, etc., are problematic regardless of thenature of the target recombinant molecule because not only can theydisplay an elution profile that may decrease purity, but they alsoremain bound to the column due to the stringent conditions necessary fortheir desorption.

The separatome-based protein expression and purification platformdisclosed herein provides the benefits of, but is not be limited to,reduction of the chromatography regimen, column capacity loss due tohost cell contaminating peptide, polypeptide, and protein adsorption,and complexity of elution protocols since the number, and nature, ofinterfering peptides, polypeptides, and proteins to be resolved is less.

The present separatome-based protein expression and purificationplatform facilitates the modification of unoptimized host cell lines inorder to eliminate the expression of undesirable, interfering peptides,polypeptides, and proteins during host cell cultivation, therebyreducing the total amount and cost of purification needed to produce ahigher concentration, and absolute amount, of purified targetrecombinant product.

The separatome-based invention disclosed herein further provides aproteomics-based protein expression and purification platform based on acomputer database and modeling system of separatome data forindividually customized cell lines that facilitate recovery andpurification of difficult to express, low yield proteins.

The separatome-based expression and purification platform disclosedherein also provides for modified host cell lines having a genomeencoding and/or expressing a reduced number of nuisance or contaminatingproteins, thereby decreasing the complexity and costs of thepurification process.

Furthermore, the present invention provides a separatome-basedexpression and purification platform that utilizes an engineered seriesof broadly applicable bacterial and other host cells to provide facilepurification systems for target recombinant peptide, polypeptide, andprotein separation.

Compared to previous approaches involving the deletion of large numbersof host cell genes, the separatome-based method for designing host cellsfor expression of target peptides, polypeptides, and proteins providedherein is more “surgical”, i.e., targeted and precise, and does notresult in the deletion of large regions of host cell genomes. Thepresent invention provides a rational framework for optimizing targetrecombinant peptide, polypeptide, or protein recovery and purificationbased on identification of host cell peptide, polypeptide, and proteincontaminants that reduce the separation efficiency, i.e., separationcapacity (product recovery), separation selectivity (product purity), orboth, of target recombinant peptides, polypeptides, and proteins basedon knowledge of the binding characteristics of contaminating speciesduring chromatographic purification. This permits the coordinated designof universally useful, optimized host cells for target recombinantpeptide, polypeptide, or protein expression and concomitant purificationprocedures using the smallest number of operations, and eliminates theneed for arbitrary, tedious, time-consuming, and expensive trial anderror experimentation. The methods disclosed herein avoid the need todesign individualized host cell expression and chromatographic systemsfor specific recombinant target proteinaceous products, and provide arational “separatomic” procedure and materials to eliminate and separatethe main interfering peptide, polypeptide, and protein components ofhost cells using the minimum number of process steps. The presentmethods and host cells minimize, or in most cases, completely avoid theproblems of eliminating host cell genes and proteins required forgrowth, viability, and target molecule expression that would adverselyaffect the use of such cells for expression of target recombinantpeptides, polypeptides, and proteins. In some cases, the presentengineered host cells exhibit improved growth, viability, and expressioncompared to the parental cells from which they are derived. This can beattributed, at least in part, to avoiding or circumventing the problemof eliminating genes that are dispensable individually, but not incombination.

SUMMARY

Accordingly, among its many embodiments, the present invention providesa separatome-based protein expression and purification platformcomprising a system of separatome data for a host cell, which comprisesdata compiled on the genome and proteome sequences of the host cell, anda data visualization tool for graphically displaying such separatomedata for identification and/or modification of contiguous or individualregions of nuisance or coeluting proteins of host cells. The separatomedata can comprise data compiled on the metalloproteome and metabolome ofthe host cell. Host cells included in this platform include, forexample, Escherichia coli, yeasts, Bacillus subtilis and otherprokaryotes, and any of the other host cells conventionally used forexpression of peptides, polypeptides, and proteins disclosed herein.

The system of separatome data is based on identified, conserved genomicregions of host cells that span resin- and gradient-specificchromatographies based on a relationship of binding properties of thepeptides, polypeptides, and proteins encoded by the identified,conserved genomic regions for these chromatographies with thecharacteristics and location of genes on the chromosome(s) of hostcells. The chromatographies include Immobilized-Metal AffinityChromatography (IMAC), cation exchange chromatography (cation TEX),anion exchange chromatography (anion IEX), Hydrophobic InteractionChromatography (HIC), or combinations thereof.

Among its many embodiments, the present invention also encompasses aseparatome-based protein expression and purification process formanufacturing of a modified cell line having a genome encoding a reducednumber of contaminating peptides, polypeptides and proteins, wherein theprocess comprises the steps of:

(1) graphically displaying a separatome of a target host cell line as avisualization tool in conjunction with relevant biochemical information;

(2) identifying specific genes, and combinations of genes, coding forcontaminating peptides, polypeptides, and proteins for the target hostcell line, and/or identifying specific genes, or combinations of genes,encoding particular nuisance peptides, polypeptides, and proteins of thetarget host cell line;

(3) identifying, when possible, large contiguous genomic regions codingfor contaminating peptides, polypeptides, and proteins for the targethost cell line, and/or identifying specific genes encoding particularnuisance peptides, polypeptides, and proteins of the target host cellline;

(4) deleting the large contiguous genomic regions coding forcontaminating peptides, polypeptides, and proteins, and/or the specificgenes, or combinations of genes, encoding particular nuisance peptides,polypeptides, and proteins, of the target host cell line from the genomeof the target host cell by large scale or targeted knockout,respectively; and

(5) deleting regions encoding any contaminant peptides, polypeptides, orproteins remaining in the genome of the target host cell after step (3)by gene specific knockout and/or PCR point mutation to form the modifiedcell line.

The target host cells specifically exemplified herein are Escherichiacoli cells conventionally used for expression.

In this process, the separatome is a system of chromatographic data ofthe juxtaposition of binding properties of peptides, polypeptides, andproteins encoded by identified, conserved genomic regions forchromatography methods with the characteristics and location of genes onthe chromosome of the target host cell. The chromatographic methods ofthis process comprise Immobilized-Metal Affinity Chromatography (IMAC),cation exchange chromatography (cation IEX), anion exchangechromatography (anion IEX), Hydrophobic Interaction Chromatography(HIC), or combinations thereof.

In this process, step (1) further comprises identifying thecontaminating proteins as essential and nonessential peptides,polypeptides, and proteins of the target host cell. Coding regions(genes) for essential peptides, polypeptides, and proteins can bereintroduced into the genome of the target host cell. The process canfurther comprise the step of constructing a larger fragment homologousto the target host cell. The fragment can be linear and sequenced withessential genes, and further comprises marker selection and selectionremoval.

The present invention also provides optimized strains of Escherichiacoli modified by a separatome-based peptide, polypeptide, and proteinexpression and purification process, wherein the strain comprises agenome having (encoding) a reduced number of nuisance or coelutingpeptides, polypeptides, and proteins. The separatome-based peptide,polypeptide, and protein expression and purification process can be atwo-step purification process based on chromatotomes of combinations ofchromatographies of Escherichia coli, and the nuisance or coelutingproteins can be reduced via large scale knockout, gene specificknockout, PCR point mutation, or a combination thereof.

More particularly, in a first set of embodiments, the present inventionencompasses the following:

1. A host cell for expression of a target recombinant peptide,polypeptide, or protein, comprising:

-   -   i) a reduced genome compared to the genome in the parent cell        from which it is derived, or    -   ii) a modified genome compared to the genome in the parent cell        from which it is derived, or    -   iii) in which expression of genes is reduced or completely        inhibited compared to expression of said genes in the parent        cell from which it is derived,    -   wherein genes that are deleted, modified, or the expression of        which is reduced or completely inhibited in said host cell, code        for peptides, polypeptides, or proteins that impair the        chromatographic separation efficiency of said target recombinant        peptide, polypeptide, or protein expressed in said host cell.        2. The host cell of 1, wherein said chromatographic separation        efficiency of said target recombinant peptide, polypeptide, or        protein is improved compared to the chromatographic separation        efficiency of said target recombinant peptide, polypeptide, or        protein in the presence of peptides, polypeptides, or proteins        coded for by said genes that are deleted, modified, or the        expression of which is reduced or completely inhibited in said        host cell upon affinity or adsorption, non-affinity column        chromatography of said target recombinant peptide, polypeptide,        or protein.        3. The host cell of 2, wherein improvement of said        chromatographic separation efficiency of said target recombinant        peptide, polypeptide, or protein is in the range of from about        5% to about 35%, or from about 10% to about 20%, compared to        chromatographic separation efficiency of said target recombinant        peptide, polypeptide, or protein in the presence of peptides,        polypeptides, or proteins coded for by said genes that are        deleted, modified, or the expression of which is reduced or        completely inhibited in said host cell upon affinity or        adsorption, non-affinity column chromatography of said target        recombinant peptide, polypeptide, or protein.        4. The host cell of any one of 1-3, wherein said chromatographic        separation efficiency is independent of elution conditions under        which said target recombinant peptide, polypeptide, or protein        emerges from an affinity or adsorption, non-affinity        chromatography column as an enriched fraction.        5. The host cell of any one of 1-4, wherein deletion of said        gene is performed by homologous recombination or frame shift        mutation.        6. The host cell of any one of 1-4, wherein modification of said        genes is performed by a method selected from the group        consisting of point mutation, isozyme substitution, and        transposon mutagenesis.        7. The host cell of any one of 1-4, wherein expression of said        genes is reduced or completely inhibited by a method selected        from the group consisting of RNA silencing, antisense        oligonucleotide inhibition, and replacement of a native promoter        with a weaker promoter.        8. The host cell of any one of 1-7, which exhibits about 75% to        about 100% of the viability, growth rate, or capacity for        expression of said target recombinant peptide, polypeptide, or        protein expressed in said host cell compared to that of said        parent cell from which it is derived, or which exhibits        viability, growth rate, or capacity for expression of said        target recombinant peptide, polypeptide, or protein expressed in        said host cell greater than that of said parent cell from which        it is derived.        9. The host cell of any one of 1-8, wherein said target        recombinant peptide, polypeptide, or protein is present in a        lysate of said host cell, or is secreted by said host cell.        10. The host cell of any one of 1-9, wherein said target        recombinant peptide, polypeptide, or protein is an endogenous        peptide, polypeptide, or protein.        11. The host cell of 10, wherein said endogenous peptide,        polypeptide, or protein is selected from the group consisting of        a nuclease, a ligase, a polymerase, an RNA- or DNA-modifying        enzyme, a carbohydrate-modifying enzyme, an isomerase, a        proteolytic enzyme, and a lipolytic enzyme.        12. The host cell of any one of 1-9, wherein said target        recombinant peptide, polypeptide, or protein is a heterologous        peptide, polypeptide, or protein.        13. The host cell of 12, wherein said heterologous peptide,        polypeptide, or protein is selected from the group consisting of        an enzyme and a therapeutic peptide, polypeptide, or protein.        14. The host cell of 13, wherein said enzyme is selected from        the group consisting of a nuclease, a ligase, a polymerase, an        RNA- or DNA-modifying enzyme, a carbohydrate-modifying enzyme,        an isomerase, a proteolytic enzyme, and a lipolytic enzyme, and        said therapeutic peptide, polypeptide, or protein is selected        from the group consisting of antibody, an antibody fragment, a        vaccine, an enzyme, a growth factor, a blood clotting factor, a        hormone, a nerve factor, an interferon, an interleukin, tissue        plasminogen activator, and insulin.        15. The host cell of any one of 1-14, which is selected from the        group consisting of a bacterium, a fungus, a mammalian cell, an        insect cell, a plant cell, and a protozoal cell.        16. The host cell of 15, wherein said bacterium is E. coli, B.        subtilis, P. fluorescens, or C. glutamicum; said fungus is a        yeast selected from the group consisting of S. cerevisiae and K.        pastoris; said mammalian cell is a CHO cell or a HEK cell; said        insect cell is an S. frugiperda cell; said plant cell is a        tobacco, alfalfa, rice, tomato, or soybean cell; and said        protozoal cell is a L. tarentolae cell.        17. The host cell of 16, wherein said bacterium is E. coli.        18. The E. coli host cell of 17, wherein said parent cell from        which said E. coli host cell is derived is selected from the        group consisting of E. coli K-12, E. coli MG, E. coli BL, and E.        coli DH.        19. The host cell of 16, wherein said bacterium is B. subtilis.        20. The B. subtilis host cell of 19, wherein said parent cell        from which said B. subtilis host cell is derived is selected        from the group consisting of B. subtilis 168 and B. subtilis        BSn5.        21. The host cell of 16, wherein said S. cerevisiae and K.        pastoris are selected from the group consisting of S. cerevisiae        S288c and AWRI796, and K. pastoris CBS7435 and GS115,        respectively.        22. The host cell of 16, wherein said CHO cell is CHO-K1 and        said HEK cell is HEK 293.        23. The E. coli parent cell of 18, which is selected from the        group consisting of E. coli K-12, E. coli MG1655, E. coli BL21        (DE3), and E. coli DH10B.        24. E. coli strain K-12. MG1655, BL21 (DE3), and DH10B of 23,        having a genome comprising the nucleotide sequence disclosed in        the reference of Table Entry Number 1, 2, 3, and 4,        respectively, in Table 1.        25. B. subtilis strain 168 and BSn5 of 20, having a genome        comprising the nucleotide sequence disclosed in the reference of        Table Entry Number 1 and 2, respectively, in Table 2.        26. S. cerevisiae strain S288c and AWR1796 of 21, having a        genome comprising the nucleotide sequence disclosed in the        reference of Table Entry Number 1 and 2, respectively, in Table        3.        27. K. pastoris strain CBS7435 and GS115 of 21, having a genome        comprising the nucleotide sequence disclosed in the reference of        Table Entry Number 1 and 2, respectively, in Table 4.        28. CHO cell strain CHOK1 of 22, having a genome comprising the        nucleotide sequence disclosed in the reference of Table Entry        Number 1 in Table 5.        29. HEK cell strain HEK 293 of 22, having a genome comprising        the nucleotide sequence disclosed in the reference of Table        Entry Number 1 in Table 6.        30. The E. coli host cell of any one of 16-18 or 23-24, wherein        said reduced genome compared to the genome in the parent cell        from which it is derived is less than 5% smaller, less than        about 4.5% smaller, less than about 4% smaller, less than about        3.5% smaller, less than about 3% smaller, less than about 2.5%        smaller, less than about 2% smaller, less than about 1.5%        smaller, or less than about 1% smaller, than the genome of said        parent cell from which it is derived.        31. The E. coli host cell of any one of 16-18 or 23-24, wherein        said reduced genome compared to the genome in the parent cell        from which it is derived is between about 4.17 Mb to about 4.346        Mb.        32. An E. coli host cell for expression of a target recombinant        peptide, polypeptide, or protein, comprising:    -   i) a reduced genome compared to the genome in the parent cell        from which it is derived, or    -   ii) a modified genome compared to the genome in the parent cell        from which it is derived, or    -   iii) in which expression of genes is reduced or completely        inhibited compared to expression of said genes in the parent        cell from which it is derived,    -   wherein said parent cell is E. coli strain K-12, MG1655. BL21        (DE3), or DH10B, having a genome comprising the nucleotide        sequence disclosed in the reference of Table Entry Number 1, 2,        3, and 4, respectively, in Table 1, and    -   wherein genes that are deleted, modified, or the expression of        which is reduced or completely inhibited in said host cell        compared to expression of said genes in said parent cell from        which it is derived, code for proteins that impair the        chromatographic separation efficiency of said target recombinant        peptide, polypeptide, or protein expressed in said host cell in        the presence of peptides, polypeptides, or proteins coded for by        said genes that are deleted, modified, or the expression of        which is reduced or completely inhibited in said host cell, and        that elute from a chromatographic affinity column having a        ligand, in a buffer comprising a compound that dictates        adsorption to its respective ligand during equilibration and        elution from said affinity column, in an amount in the range, in        a combination selected from the group consisting of the        combinations in the following table:

Compound in Buffer That Dictates Adsorption to Affinity Column DuringEquilibration and Causes Ligand Elution From Column Concentration or pHRange Glutathione S- Glutathione from about 0 mM to about 10 mMtransferase Amino acid A common salt from about 0 mM to about 2M (e.g.,lysine) Amino acid pH from about pH 2 to about pH 11 Avidin A chaotropicsalt from about 0M to about 4M Avidin pH from about pH 2 to about pH10.5 Carbohydrate Sugar or isocratic from about 0 mM to about 10 mM(e.g., Dextrin) (e.g., maltose) Carbohydrate pH from about pH 5 to aboutpH 8 Organic dye A common salt from about 0 mM to about 1.5M (e.g.,Cibacron Blue) Organic dye pH from about pH 4 to about pH 8 Organic dyeImidazole from about 5 mM to about 250 mM or a common salt Divalentmetal pH from about pH 4 to about pH 12 (e.g., Ni(II)) Divalent metalImidazole from about 5 mM to about 500 mM (e.g., Ni(II)) Heparin Acommon salt from about 0 mM to about 2M Protein A or Protein G Glycinefrom about 0 mM to about 100 mM Protein A or Protein G pH from about pH3 to about pH 7 IgG Glycine from about 0 mM to about 100 mM CoenzymeCompeting Protein from about 1 mM to about 12 mM33. An E. coli host cell for expression of a target recombinant peptide,polypeptide, or protein, comprising:

-   -   i) a reduced genome compared to the genome in the parent cell        from which it is derived, or    -   ii) a modified genome compared to the genome in the parent cell        from which it is derived, or    -   iii) in which expression of genes is reduced or completely        inhibited compared to expression of said genes in the parent        cell from which it is derived,    -   wherein said parent cell is E. coli strain K-12, MG1655, BL21        (DE3), or DH10B, having a genome comprising the nucleotide        sequence disclosed in the reference of Table Entry Number 1, 2,        3, and 4, respectively, in Table 1,    -   wherein genes that are deleted, modified, or the expression of        which is reduced or completely inhibited in said host cell, code        for host cell peptides, polypeptides, or proteins that impair        the chromatographic separation efficiency of said target        recombinant peptide, polypeptide, or protein expressed in said        host cell, and    -   wherein genes that are deleted, modified, or the expression of        which is reduced or completely inhibited in said host cell        compared to expression of said genes in said parent cell from        which it is derived, code for proteins that impair the        chromatographic separation efficiency of said target recombinant        peptide, polypeptide, or protein expressed in said host cell in        the presence of peptides, polypeptides, or proteins coded for by        said genes that are deleted, modified, or the expression of        which is reduced or completely inhibited in said host cell, and        that elute from a chromatographic adsorption, non-affinity        column having a ligand, in a buffer comprising a compound that        dictates adsorption to its respective ligand during        equilibration and elution from said adsorption, non-affinity        column, in an amount in the range, in a combination selected        from the group consisting of the combinations in the following        table:

Compound in Buffer That Dictates Adsorption to Non-Affinity ColumnDuring Equilibration and Causes Elution From Ligand Column Concentrationor pH Range Ion exchange Common salt from about 0M to about 2M Ionexchange pH from about pH 2 to about pH 12 Reverse phase Organic solventex. from about 0% to about 100% Acetonitrile Hydrophobic Common saltfrom about 2M to about 0M interaction34. The E. coli host cell of 32 or 33, wherein said common salt isselected from the group consisting of a chloride salt, a sulfate salt,an acetate salt, a carbonate salt, and a propionate salt.35. The E. coli host cell of 33, wherein said organic solvent isselected from the group consisting of acetonitrile, methanol, and2-propanol.36. The E. coli host cell of 33, wherein genes that are deleted,modified, or the expression of which is inhibited, in the genome of saidE. coli host cell are selected from the group consisting of:

GeneName rpoC rpoB hldD metH entF mukB tgt rnr glgP recC ycaO glnA ptsImetE sucA hrpA groL gatZ speA thiI nusA tufA degP clpB rapA metL ycfDnagD ilvA fusA cyaA gldA dnaK ygiC gyrA glnE carB ppsA degQ usg ilvBthrS recB entB dusA typA prs cysN atpD purLand combinations thereof.37. The E. coli host cell of 33, wherein said parent cell E. coli strainis MG1655 (genotype: Wild Type: F−, λ⁻, rph-1), and the followingcombinations of genes are deleted, modified, or the expression of whichis inhibited: LTS00 (genotype: ΔthyA); LTS01+ (genotype: ΔmetH); LTS01(genotype: ΔthyAΔmetH); LTS02+ (genotype: ΔmetHΔentF); LTS02 (genotype:ΔthyAΔmetHΔentF); LTS03+ (genotype: ΔmetHΔentFΔtgt); LTS03 (genotype:ΔthyAΔmetHΔentFΔtgt); LTS04+ (genotype: ΔmetHΔentFΔtgtΔrnr); LTS04(genotype: ΔthyAΔmetHΔentFΔtgtΔrnr); or LTS05+ (genotype:ΔmetHΔentFΔtgtΔrnrΔycaO).38. The host cell of any one of 1-37, wherein increased separationefficiency is manifested as increased separation capacity, increasedseparation selectivity, or both.39. The host cell of 38, wherein separation capacity is defined as theamount of target recombinant peptide, polypeptide, or protein adsorbedto said column per mass lysate in the case where said target recombinantpeptide, polypeptide, or protein is not secreted, or mass culture mediumin the case where said target recombinant peptide, polypeptide, orprotein is secreted, applied to said column, and separation selectivityis defined as the amount of target recombinant peptide, polypeptide, orprotein adsorbed to said column per total peptide, polypeptide, orprotein adsorbed to said column.40. The host cell of 38 or 39, wherein said increased separationcapacity is in the range of from about 5% to about 35%.41. The host cell of any one of 1-40, wherein separation of said targetrecombinant peptide, polypeptide, or protein from host cell peptides,polypeptides, or proteins is performed by column chromatographyemploying a solid phase chromatography medium.42. The host cell of 41, wherein said column chromatography is selectedfrom the group consisting of affinity chromatography employing anaffinity ligand bound to said solid phase, and adsorption-based,non-affinity chromatography.43. The host cell of 42, wherein said affinity ligand is selected fromthe group consisting of an amino acid, a divalent metal ion, acarbohydrate, an organic dye, a coenzyme; glutathione S-transferase,avidin, heparin, protein A, and protein G.44. The host cell of 43, wherein said divalent metal ion is selectedfrom the group consisting of Cu⁺⁺, Ni⁺⁺, Co⁺⁺, and Zn⁺⁺; saidcarbohydrate is selected from the group consisting of maltose,arabinose, and glucose; said organic dye is a dye comprising a triazenemoiety; and said coenzyme is selected from the group consisting of NADHand ATP.45. The host cell of 42, wherein said adsorption-based, non-affinitychromatography is selected from the group consisting of ion exchangechromatography, reverse phase chromatography, and hydrophobicinteraction chromatography.46. The host cell of 45, wherein said adsorption-based, non-affinitychromatography is ion exchange chromatography.47. The host cell of 46, wherein said ion exchange chromatographyemploys a ligand selected from the group consisting of diethylaminoethylcellulose (DEAE), monoQ, and S.48. The host cell of any one of 41 to 47, wherein said host cellpeptides, polypeptides, or proteins that impair separation efficiency ofsaid target recombinant peptide, polypeptide, or protein expressed insaid host cell are peptides, polypeptides, or proteins that are stronglyretained during column chromatography.49. The host cell of 48, wherein said host cell peptides, polypeptides,or proteins that are strongly retained during ion exchangechromatography are those that are retained during elution with a mobilephase comprising a common salt in the range of from about 5 mM to about2,000 mM.50. The host cell of 49, wherein said host cell peptides, polypeptides,or proteins that are strongly retained during ion exchangechromatography are those that are retained during elution with a mobilephase comprising a common salt in the range of from about 500 mM toabout 1,000 mM.51. The host cell of any one of 41 to 50, wherein said host cellpeptides, polypeptides, or proteins that impair the separationefficiency of said target recombinant peptide, polypeptide, or proteinexpressed in said host cell are peptides, polypeptides, or proteins thatare weakly retained during column chromatography.52. The host cell of 50, wherein said host cell peptides, polypeptides,or proteins that are weakly retained during chromatography are thosethat are retained during elution with a mobile phase comprising a commonsalt in the range of from about 5 mM to about 500 mM.53. The host cell of 52, wherein said host cell peptides, polypeptides,or proteins that are weakly retained during chromatography are thosethat are retained during elution with a mobile phase comprising a commonsalt in the range of from about 10 mM to about 350 mM.54. The host cell of any one of 41 to 53, wherein said host cellpeptides, polypeptides, or proteins that impair the separationefficiency of said target recombinant peptide, polypeptide, or proteinexpressed in said host cell are peptides, polypeptides, or proteins thatare both strongly retained and weakly retained during columnchromatography.55. A separatome of chromatographically relevant host cell peptides,polypeptides, and proteins for column affinity chromatography employingan affinity ligand bound to a solid phase or column adsorption-based,non-affinity chromatography, comprising host cell peptides,polypeptides, and proteins based on their capacity recovery potentialfrom said column.

-   -   wherein said capacity recovery potential of said host cell        peptides, polypeptides, and proteins is quantitatively        determined by:    -   (a) scoring a peptide, polypeptide, or protein (i) with the        formulae:

$\begin{matrix}{{importance}_{i} = {\Sigma_{j}\left\lbrack {{b_{1}\left( \frac{y_{c_{j}}}{y_{\max}} \right)}\left( \frac{h_{i,_{j}}}{h_{i,{total}}} \right)\left( \frac{h_{i,_{j}}}{h_{j,{total}}} \right)\left( \frac{{MW}_{i}}{{MW}_{ref}} \right)^{\alpha}} \right\rbrack}_{i}} & {{Equation}\mspace{14mu} 3}\end{matrix}$

with values for a series of peptides, polypeptides, and proteins writtenin descending order (largest value close to unity downwards to thesmallest value), followed by

-   -   (b) calculating the capacity recovery potential of a relevant        peptide, polypeptide, or protein (i) given by:

recovery potential_(i) =h _(i,total) /h _(total,ms),  Equation 1

-   -   wherein the following definitions apply: y_(cj) and        y_(max)=concentration of mobile phase eluent in fraction (j) and        maximum value, respectively; and h_(i,j) and h_(i,total)=the        amount of protein (i) in fraction (j) and total bound protein        (i), respectively; h_(j,total)=total amount of protein in        fraction (j); h_(total,ms)=total mass of protein bound to        column; b₁=scaling parameter; α=steric factor; MW_(j) and        MW_(ref)=molecular weight of protein (i) or reference protein,        respectively.        56. The separatome of 55, wherein said affinity ligand in said        column affinity chromatography employing an affinity ligand        bound to a solid phase is selected from the group consisting of        an amino acid, a divalent metal ion, a carbohydrate, an organic        dye, a coenzyme, glutathione S-transferase, avidin, heparin,        protein A, and protein G.        57. The separatome of 56, wherein peptides, polypeptides, and        proteins are eluted from said affinity chromatography column        using an elution agent y selected from the group consisting of a        common salt, hydronium ion, imidazole, glutathione, a chaotropic        salt, heparin, and glycine.        58. The separatome of 55, wherein said column adsorption-based,        non-affinity chromatography is selected from the group        consisting of ion exchange chromatography, reverse phase        chromatography, and hydrophobic interaction chromatography.        59. The separatome of 58, wherein peptides, polypeptides, and        proteins are eluted from said adsorption-based, non-affinity        chromatography column using an elution agent y selected from the        group consisting of a common salt, hydronium ion, and an organic        solvent.        60. The separatome of 57 or 59, wherein said common salt is        selected from the group consisting of a chloride salt, a sulfate        salt, an acetate salt, a carbonate salt, and a propionate salt.        61. The separatome of 59, wherein said organic solvent is        selected from the group consisting of methanol, 2-propanol, and        acetonitrile.        62. The separatome of 57, wherein said chaotropic salt is        guanidine hydrochloride.        63. The separatome of any one of 55-62, wherein the maximum        value of said elution agent y is defined by y_(max) in 55.        64. The separatome of any one of 55-63, which is in a form        selected from the group consisting of a table, a visual        representation such as a figure, and a computer file.        65. The separatome of chromatographically relevant host cell        peptides, polypeptides, or proteins for column affinity        chromatography employing an affinity ligand bound to a solid        phase of any one of 55-57, 60, or 62-64.        66. The separatome of chromatographically relevant host cell        peptides, polypeptides, or proteins for column adsorption-based,        non-affinity chromatography of any one of 55, 58-61, or 63-64.        67. A method for designing a reduced or modified proteome host        cell, or a host cell in which expression of genes is reduced or        completely inhibited compared to expression of said genes in the        parent cell from which said host cell is derived, for expression        of a target recombinant peptide, polypeptide, or protein to        improve the chromatographic separation efficiency of said target        recombinant peptide, polypeptide, or protein expressed in said        host cell, comprising identifying and ranking proteins of        chromatographic relevance that adversely affect said separation        efficiency of said target recombinant peptide, polypeptide, or        protein in a parent cell from which said host cell is derived        by:    -   i) equilibrating an affinity chromatography column employing an        affinity ligand bound to a solid phase, or an adsorption-based,        non-affinity chromatography column, using a mobile loading or        eluting phase, or an operational variable;    -   ii) in the case where said target recombinant peptide,        polypeptide, or protein is not secreted, fractionating a lysate        of said host cell, or in the case where said target recombinant        peptide, polypeptide, or protein is secreted from said host        cell, fractionating the culture medium in which said host cell        is grown, on said column by applying an elution gradient to        elute peptide, polypeptide, or protein fractions from said        column;    -   iii) identifying, quantifying, and scoring peptides,        polypeptides, or proteins in said fractions eluted from said        column;    -   iv) assessing the metabolic role of said peptides, polypeptides,        or proteins identified in step iii) that affect column capacity;        and    -   v) designing a reduced or modified genome host cell, or a host        cell in which expression of genes is reduced or completely        inhibited compared to expression of said genes in the parent        cell from which said host cell is derived, to modify the        proteome of said parent cell from which said host cell is        derived in order to increase chromatographic separation        efficiency based on steps iii) and iv).        68. The method of 67, further comprising reducing or modifying        the genome of said parent cell from which said host cell is        derived, or reducing or completely inhibiting the expression of        peptides, polypeptides, or proteins in said parent cell, to        increase chromatographic separation efficiency based on step v),        thereby producing a host cell comprising a reduced or modified        genome compared to the genome in said parent cell from which        said host cell is derived, or a host cell in which expression of        peptides, polypeptides, or proteins is reduced or completely        inhibited.        69. The method of 67 or 68, wherein said reduced or modified        proteome host cell, or said host cell host cell in which        expression of (n) genes is reduced or completely inhibited        compared to expression of said genes in the parent cell from        which said host cell is derived, facilitates an overall capacity        recovery of said target recombinant peptide, polypeptide, or        protein in the range of from about 5%, from about 10%, from        about 20%, from about 30%, from about 40%, from about 50%, from        about 60%, from about 70%, from about 80%, from about 90%, or        from about 95%, to about 100%, wherein capacity recovery is        defined by summing (n) values of recovery potential for        individual (i) proteins by the following:

$\begin{matrix}{{{capacity}\mspace{14mu} {recovery}} = {100\% \mspace{11mu} x{\sum\limits_{i = 1}^{n}\; {{recovery}\mspace{14mu} {potential}_{i}}}}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

-   -   wherein n=total number of proteins that are deleted, inhibited,        or modified, and i=an individual protein.

A preferred range for capacity recovery is from about 3% to about 50%,more preferably from about 5% to about 40%, or from about 5% to about35%,

70. The method of any one of 67-69, wherein step i) is modified byvarying the characteristics of said mobile loading or eluting phase oroperational variable.71. The method of any one of 67-70, wherein identification of saidpeptides, polypeptides, or proteins in step iii) is performed bycomparing the LC-MS signature of said peptides, polypeptides, orproteins to publicly available standards.72. The method of any one of 67-71, wherein quantification of saidproteins in step iii) is performed using spectral counting, or acombination of Bradford protein assay, 2-dimensional electrophoresis,and densitometry.73. The method of any one of 67-72, wherein said scoring in step iii) iscalculated as in 55.74. The method of any one of 67-73, wherein assessing the metabolic roleof identified proteins in step iv) is performed by bioinformaticstechniques.75. A method of enriching the amount of a target recombinant peptide,polypeptide, or protein relative to other peptides, polypeptides, orproteins present in an initial protein mixture comprising said targetrecombinant peptide, polypeptide, or protein, comprising:

-   -   i) selecting a chromatography medium that binds said target        recombinant peptide, polypeptide, or protein from the group        consisting of an affinity chromatography medium and an        adsorption-based, non-affinity chromatography medium;    -   ii) in the case where an affinity chromatography medium is        selected, expressing said target recombinant peptide,        polypeptide, or protein in said host cell of any one of 1-32,        34, 36, 38-44, 48, or 51-54;    -   iii) in the case where an adsorption-based, non-affinity        chromatography medium is selected, expressing said target        recombinant peptide, polypeptide, or protein in said host cell        of any one of 1-31, 33-35, 37-42, or 45-54; and    -   iv) chromatographing said initial protein mixture comprising        said target recombinant peptide, polypeptide, or protein using        said chromatography medium of step ii) or step iii), as        appropriate, and collecting elution fractions, thereby obtaining        one or more fractions containing an enriched amount of said        target recombinant peptide, polypeptide, or protein relative to        other peptides, polypeptides, or proteins in said fraction        compared to the amount of said target recombinant peptide,        polypeptide, or protein relative to other peptides,        polypeptides, or proteins in said initial protein mixture.        76. The method of 75, further comprising chromatographing an        enriched fraction of step iv) to obtain said target recombinant        peptide, polypeptide, or protein in a desired degree of purity.        77. The method of 76, further comprising recovering said target        recombinant peptide, polypeptide, or protein.        78. A method of preparing a pharmaceutical or veterinary        composition comprising a recombinant therapeutic peptide,        polypeptide, or protein, comprising:    -   i) selecting a chromatography medium that binds said recombinant        therapeutic peptide, polypeptide, or protein from the group        consisting of an affinity chromatography medium and an        adsorption-based, non-affinity chromatography medium;    -   ii) in the case where an affinity chromatography medium is        selected, expressing said recombinant therapeutic peptide,        polypeptide, or protein in said host cell of any one of 1-32,        34, 36, 38-44, 48, or 51-54;    -   iii) in the case where an adsorption-based, non-affinity        chromatography medium is selected, expressing said recombinant        therapeutic peptide, polypeptide, or protein in said host cell        of any one of 1-31, 33-35, 3742, or 45-54;    -   iv) in the case where said recombinant therapeutic peptide,        polypeptide, or protein is not secreted from said host cell,        preparing a lysate of said host cell containing said recombinant        therapeutic peptide, polypeptide, or protein, producing an        initial recombinant therapeutic peptide-, polypeptide-, or        protein-containing mixture; or    -   v) in the case where said recombinant therapeutic peptide,        polypeptide, or protein is secreted from said host cell,        harvesting culture medium in which said host cell is grown,        containing said recombinant therapeutic peptide, polypeptide, or        protein, thereby obtaining an initial recombinant therapeutic        peptide-, polypeptide-, or protein-containing mixture;    -   vi) chromatographing said initial recombinant therapeutic        peptide-, polypeptide-, or protein-containing mixture of        step iv) or step v) using said chromatography medium of step i)        or step ii), as appropriate, and collecting elution fractions,        thereby obtaining one or more fractions containing an enriched        amount of said recombinant therapeutic peptide, polypeptide, or        protein relative to other peptides, polypeptides, or proteins in        said fraction compared to the amount of said recombinant        therapeutic peptide, polypeptide, or protein relative to other        peptides, polypeptides, or proteins in said initial protein        mixture;    -   vii) further chromatographing an enriched fraction of step vi)        to obtain said recombinant peptide, polypeptide, or protein in a        desired degree of purity;    -   viii) recovering said recombinant therapeutic peptide,        polypeptide, or protein; and    -   ix) formulating said recombinant therapeutic peptide,        polypeptide, or protein with a pharmaceutically or veterinarily        acceptable carrier, diluent, or excipient to produce a        pharmaceutical or veterinary composition, respectively.        79. A method of purifying a recombinant enzyme, comprising:    -   i) selecting a chromatography medium that binds said recombinant        enzyme from the group consisting of an affinity chromatography        medium and an adsorption-based, non-affinity chromatography        medium;    -   ii) in the case where an affinity chromatography medium is        selected, expressing said recombinant enzyme in said host cell        of any one of 1-32, 34, 36, 38-44, 48, or 51-54;    -   iii) in the case where an adsorption-based, non-affinity        chromatography medium is selected, expressing said recombinant        enzyme in said host cell of any one of 1-31, 33-35, 37-42, or        45-54;    -   iv) in the case where said recombinant enzyme is not secreted        from said host cell, preparing a lysate of said host cell        containing said recombinant enzyme, producing an initial        recombinant enzyme-containing mixture; or    -   v) in the case where said recombinant enzyme is secreted from        said host cell, harvesting culture medium in which said host        cell is grown, containing said recombinant enzyme, thereby        obtaining an initial recombinant enzyme-containing mixture;    -   vi) chromatographing said initial recombinant enzyme-containing        mixture of step iv) or step v) using said chromatographic medium        of step i) or step ii), as appropriate, and collecting elution        fractions, thereby obtaining one or more fractions containing an        enriched amount of said recombinant enzyme relative to other        peptides, polypeptides, or proteins in said fraction compared to        the amount of said recombinant enzyme relative to other        peptides, polypeptides, or proteins in said initial protein        mixture;    -   vii) further chromatographing an enriched fraction of step vi)        to obtain said recombinant enzyme in a desired degree of purity;        and    -   viii) recovering purified recombinant enzyme.        80. The method of 79, further comprising placing said purified        recombinant enzyme in a buffer solution in which said purified        recombinant enzyme is stable and retains enzymatic activity.        81. The method of 80, wherein said purified recombinant        enzyme-containing buffer solution is reduced to dryness.        82. The method of 81, wherein said dry purified recombinant        enzyme-containing buffer solution is in the form of a powder.        83. A kit, comprising said host cell of any one of 1-54 or        68-69.        84. The kit of 83, further comprising instructions for        expressing a target recombinant peptide, polypeptide, or protein        in said host cell.        85. The kit of 84, wherein said target recombinant peptide,        polypeptide, or protein is an endogenous or heterologous target        recombinant peptide, polypeptide, or protein.        86. The kit of any one of 83-85, wherein said instructions        further comprise directions for purifying said expressed target        recombinant peptide, polypeptide, or protein by affinity        chromatography or adsorption-based, non-affinity chromatography.        87. The kit of any one of 83-86, further comprising a        chromatographic resin for affinity chromatography or        adsorption-based, non-affinity chromatography.        88. A method of enriching a target peptide, polypeptide, or        protein from a mixture obtained from a host cell, comprising:    -   a. chromatographing said mixture via affinity chromatography or        adsorption-based, non-affinity chromatography;    -   b. collecting an elution fraction that contains an enriched        amount of said target peptide, polypeptide, or protein in said        fraction compared to the amount of said peptide, polypeptide, or        protein of interest in said mixture; and    -   c. recovering said target peptide, polypeptide, or protein from        said elution fraction, wherein said host cell is derived from a        parent cell, and has:        -   i) a reduced genome compared to the genome in the parent            cell from which it is derived, or        -   ii) a modified genome compared to the genome in the parent            cell from which it is derived, or        -   iii) in which expression of genes is reduced or completely            inhibited compared to expression of said genes in the parent            cell from which it is derived,        -   wherein genes that are deleted, modified, or the expression            of which is reduced or completely inhibited in said host            cell, code for peptides, polypeptides, or proteins that            impair the chromatographic separation efficiency of said            target peptide, polypeptide, or protein expressed in said            host cell in said affinity chromatography or said            adsorption-based, non-affinity chromatography.            89. The method of 88, wherein said mixture is a lysate of            said host cell in the case where said peptide, polypeptide,            or protein accumulates intracellularly, or is medium in            which said host cell is grown in the case where said            peptide, polypeptide, or protein is secreted by said host            cell.            90. The method of 88 or 89, further comprising            chromatographing said target peptide, polypeptide, or            protein of step c, in order to obtain said target peptide,            polypeptide, or protein in a desired degree of purity.            91. The method of 90, further comprising recovering purified            target peptide, polypeptide, or protein.

The methods of 88-91 encompass the use of all of the parent cells, hostcells, and methods, etc., disclosed herein, and described in 1-87,above.

In a second set of embodiments, the present invention encompasses thefollowing:

1. An E. coli host cell, derived from a parent E. coli cell, forexpression of a target host cell or target recombinant peptide,polypeptide, or protein, said E. coli host cell comprising:

-   -   i) a reduced genome compared to the genome in the parent cell        from which it is derived, and/or    -   ii) a modified genome compared to the genome in the parent cell        from which it is derived, and/or    -   iii) in which expression of genes is reduced or completely        inhibited compared to expression of said genes in the parent        cell from which it is derived,    -   wherein genes that are deleted, modified, and/or the expression        of which is reduced or completely inhibited in said host cell        code for peptides, polypeptides, or proteins that impair the        chromatographic separation efficiency of said target host cell        or target recombinant peptide, polypeptide, or protein expressed        in said host cell,    -   wherein said genes are selected from the group consisting of:    -   the genes listed in Table 8, and combinations thereof;    -   the genes listed in Table 9, and combinations thereof;    -   combinations of any of the genes listed in Tables 8 and 9 taken        together;    -   the genes listed in Table 14, and combinations thereof; and    -   combinations of any of the genes listed in Tables 8, 9, and 14        taken together.        2. The E. coli host cell of 1, wherein said parent cell is an E.        coli strain selected from the group consisting of strain K-12,        strain B, strain C, strain W, and a derivative of any of the        foregoing strains.        3. The E. coli host cell of 2, wherein:    -   said E. coli strain K-12 derivative is selected from the group        consisting of W3110, DH10B, DH5alpha, DH1, MG1655, and BW2952;        and    -   said E. coli strain B derivative is selected from the group        consisting of B REL606, BL21, and BL21-DE3.        4. The E. coli host cell of any one of 1-3, wherein said        parent E. coli cell is selected from the group consisting of:    -   Alpha-Select Bacteriophage T1-Resistant Gold Efficiency (F− deoR        endA1 recA1 relA1 gyrA96 hsdR17(rk⁻, mk₊) supE44 thi-1 phoA        Δ(lacZYA-argF)U169 Φ80lacZΔM15λ−),    -   Alpha-Select Bacteriophage T1-Resistant Silver Efficiency (F−        deoR endA1 recA1 relA1 gyrA96 hsdR17(rk⁻, mk₊) supE44 thi-1 phoA        Δ(lacZYA-argF)U169 Φ80lacZΔM15λ−),    -   Alpha-Select Bronze Efficiency (F− deoR endA1 recA1 relA1 gyrA96        hsdR17(rk−, mk+) supE44 thi-1 phoA Δ(lacZYA-argF)U169        Φ80lacZΔM15λ−),    -   Alpha-Select (F− deoR endA1 recA1 relA1 gyrA96 hsdR17(rk−, mk+)        supE44 thi-1 phoA Δ(lacZYA-argF)U169 Φ80lacZΔM15λ−),    -   AG1 (endA1 recA1 gyrA96 thi-1 relA1 glnV44 hsdR17(r_(K) ⁻ m_(K)        ⁺)),    -   AB1157 (thr-1, araC14, leuB6(Am), Δ(gpt-proA)62, lacY1, tsx-33,        qsr′-0, glnV44(AS), galK2(Oc), LAM−, Rac-0, hisG4(Oc), rfbC1,        mgl-51, rpoS396(Am), rpsL31(strR), kdgK51, xylA5, mtl-1,        argE3(Oc), thi-1),    -   B2155 (thrB1004 pro thi strA hsdsS lacZD M15 (F′lacZD M15        lacI^(q) traD36 proA⁺proB⁺) Δ dapA::erm (Erm^(r)) pir::RP4        [::kan (Km^(r)) from SM10]),    -   B834(DE3) (F⁻ompT hsdS_(B)(r_(B) ⁻ m_(B) ⁻) gal dcm met (DE3)),    -   BIOBlue (recA1 endA1 gyrA96 thi-1 hsdR17(rk−, mk+) supE44 relA1        lac [F′ proAB lacI^(q)ZΔM15 Tn10(Tet^(r))]),    -   BL21 (E. coli B F− dcm ompT hsdS(r_(B)− m_(B)−) gal        [malB⁺]_(K-12)(λ^(S))),    -   BL21(AI) (F⁻ompT gal dcm lon hsdS_(B)(r_(B) ⁻ m_(B) ⁻)        araB::T7RNAP-tetA),    -   BL21(DE3) (F⁻ ompT gal dcm lon hsdS_(B)(r_(B) ⁻ m_(B) ⁻) λ(DE3        [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])),    -   BL21 (DE3) pLysS (F− ompT hsdSB(rB−, mB−) gal dcm (DE3) pLysS        (CamR)),    -   BL21-T1R (F− ompT hsdSB(rB− mB−) gal dcm tonA),    -   BNN93 (F⁻ tonA21 thi-1 thr-1 leuB6 lacY1 glnV44 rfbC1 fhuA1 mcrB        e14-(mcrA⁻) hsdR(r_(K) ⁻m_(K) ⁺) λ⁻)    -   BNN97 (BNN93 (λgt11)),    -   BW26434 (Δ(araD-araB)567, Δ(lacA-lacZ)514(::kan),        lacI^(p)-4000(lacI^(q)), λ⁻, rpoS396(Am)?, rph-1,        Δ(rhaD-rhaB)568, bsdR514),    -   C600 (F⁻ tonA21 thi-1 thr-1 leuB6 lacY1 glnV44 rfbC1 fhuA1λ⁻),    -   CAG597 (F⁻ lacZ(am) pho(am) lyrT[supC(ts)] trp(am) rpsL(Str^(R))        rpoH(am)165 zhg::Tn10 mal(am)),    -   CAG626 (F⁻ lacZ(am) pho(am) lon trp(am) tyrT[supC(ts)]        rpsL(Str^(R)) mal(am)),    -   CAG629 (F⁻ lacZ(am) pho(am) lon supC(ts) trp(am) rpsL        rpoH(am)165 zhg::Tn10 mal(am)),    -   CH3-Blue (F− ΔmcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1        endA1 ara Δ139 Δ(ara, leu)7697 galU galrpsL(Str^(R)) nupG λ−).    -   CSH50 (F⁻ λ⁻ ara Δ(lac-pro) rpsL thi fimE::IS1),    -   D1210 (HB101 lacI^(q) lacY⁺),    -   dam-dcm-Bacteriophage T1-Resistant (F− dam-13:Tn9(Cam^(R))dcm-6        ara-14 hisG4 leuB6 thi-1 lacY1 galK2 galT22 glnV44 hsdR2 xylA5        mtl-1 rpsL136(Str^(R)) rtbD1 tonA31 tsx78 mcrA mcrB1),    -   DB3.1 (F− gyrA462 endA1 glnV44 Δ(sr1-recA) mcrB mrr hsdS20(r_(B)        ⁻, m_(B) ⁺) ara14 galK2 lacY1 proA2 rpsL20(Sm^(r)) xy15 Δleu        mtl1),    -   DH1 (endA1 recA1 gyrA96 thi-1 glnV44 relA1 hsdR17(r_(K) ⁻ m_(K)        ⁺) λ⁻),    -   DH5α Turbo (F′ proA+B+ lacI^(q) Δ lacZ M15/fhuA2 Δ(lac-proAB)        glnV gal R(zgb-210::Tn10)Tet^(S) endA1 thi-1 Δ(hsdS-mcrB)5),    -   DH12S (mcrA Δ(mrr-hsdRMS-mcrBC) φ80d lacZΔM15 ΔlacX74 recA1 deoR        Δ(ara, leu)7697 araD139 galU galK rpsL F′ [proAB⁺        lacI^(q)ZΔM15]),    -   DM1 (F− dam-13::Tn9(Cm^(R)) dcm− mcrB hsdR-M+ gal1 gal2 ara−        lac− thr− leu− tonR tsxR Su0),    -   E. CLONI® 5ALPHA (fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80        Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17),    -   E. CLONI® 10G (F− mcrA Δ(mnr-hsdRMS-mcrBC) endA1 recA1        Φ80dlacZΔM15 ΔlacX74 araI139 Δ(ara,leu)7697galU galK rpsL nupG        λ− tonA (StrR)),    -   E. CLONI® 10GF′ ([F′ pro A+B+ lacI^(q)ZΔM15::T10 (Tet^(R))]/mcrA        Δ(mrr-hsdRMS-mcrBC) endA1 recA1 Φ80dlacZΔM15 ΔlacX74 araD139        Δ(ara, leu)7697 galU galK rpsL nupG λ− tonA (StrR)),    -   E. coli K12 ER2738 (F′proA+B+ lacI^(q) Δ(lacZ)M15        zzf::Tn10(Tet^(R))/fhuA2 glnV Δ(lac-proAB) thi-1 Δ(hsdS-mcrB)5),    -   ElectroMax™ DH10B (F⁻mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15        ΔlacX74 recA1 endA1 araD139Δ(ara,leu)7697 galU galK λ⁻rpsL        nupG),    -   ELECTROMAX™ DH5ALPHA-E (F− φ80lacZΔM15 Δ(lacZY A-argF) U169        recA1 endA1 hsdR17 (rk−, mk+) galphoA supE44λ-thi-1 gyrA96        relA1),    -   ElectroSHOX (F− mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74        recA1 endA1 ara Δ139 Δ(ara, leu)7697 galU galKrpsL(Str^(R)) nupG        λ⁻)    -   EP-MAX™10B F′ (mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74        deoR recA1 endA1 araD139 Δ(ara, leu)7697 galU galK rpsL nupG        λ−/F′[lacI^(q)ZΔM15 Tn10 (Tet^(R))]),    -   ER1793 (F⁻ fhuA2 Δ(lacZ)r1 glnV44 e14⁻(McrA⁻) trp-31 his-1        rpsL104 xyl-7 mtl-2 metB1 Δ(mcrC-mrr)114::IS10),    -   ER1821 (F⁻ glnV44 e14⁻(McrA⁻) rfbD1? rel4? endA1 spoT1? thi-1        Δ(mcrC-mrr)114::IS10),    -   ER2738 (F′proA⁺B⁺ lacI^(q) Δ(lacZ)M15 zzf::Tn10(Tet^(R))/fhuA2        glnV Δ(lac-proAB) thi-1 Δ(hsdS-mcrB)5),    -   ER2267 (F′ proA⁺B⁺ lacI^(q) Δ(lacZ)M15 zzf::mini-Tn10        (Kan^(R))/Δ(argF-lacZ)U169 glnV44 e14⁻(McrA⁻) rfbD1? recA1        relA1? endA1 spoT1? thi-1 Δ(mcrC-mrr)114::IS10),    -   ER2507 (F⁻ ara-14 leuB6 fhuA2 Δ(argF-lac)U169 lacY1 glnV44 galK2        rpsL20 xyl-5 mtl-5 Δ(malB)        zjc::Tn5(Kan^(R))Δ(mcrC-mrr)_(HB101)),    -   ER2508 (F⁻ ara-14 leuB6 fhuA2 Δ(argF-lac)U169 lacY1        lon::miniTn10(Tet^(R)) glnV44 galK2 rpsL20(Str^(R)) xyl-5 mtl-5        Δ(malB) zjc::Tn5(Kan^(R)) Δ(mcrC-mrr)_(HB101))    -   ER2738 (F′proA⁺B⁺ lacI^(q) Δ(lacZ)M15 zzf::Tn10(Tet^(R))/fhuA2        glnV Δ(lac-proAB) thi-1 Δ(hsdS-mcrB)5),    -   ER2925 (ara-14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA        dcm-6 hisG4 rfbD1 R(zgb210::Tn10)Tet^(S) endA1 rpsL136        dam13::Tn9 xylA-5 mtl-1 thi-1 mcrB1 hsdR2),    -   GC5™ (:F− Φ80lacZ Δ M15 Δ (lacZYA-argF)U169 endA1 recA1 relA1        gyrA96 hsdR17 (r_(k) ⁻, m_(k) ⁺) phoA supE44 thi-1λ−T1R),    -   GC10 (F− mcrA Δ(mrr-hsdRMSmcrBC) Φ80dlacZ Δ M15 Δ lacX74 endA1        recA1 Δ (ara, leu)7697 araD139 galUgalK nupG rpsL λ−T1R),    -   GENEHOGS® (FmcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1        araD139 Δ(araleu)7697 galU galK rpsL (StrR) endA1 nupG fhuA::IS2        (confers phage T1 resistance)),    -   HB101,    -   HMS174,    -   HMS174(DE3),    -   HI-CONTROL™ BL21(DE3) (F⁻ ompT gal dcm hsdS_(B)(r_(B) ⁻ m_(B) ⁻)        (DE3)/Mini-F lacI^(q1)(Gent^(r))).    -   HI-CONTROL™ 10G (F− mcrA Δ(mrr-hsdRMS-mcrBC) endA1 recA1        Φ80dlacZΔM15 ΔlacX74araD139 Δ(ara,leu)7697 galU galK rpsL nupG        λ− tonA/Mini-F lacI^(q1) (Gent^(r))),    -   HT96™ NOVABLUE (endA1 hsdR17 (r_(K12) ⁻ m_(K12) ⁺) supE44 thi-1        recA1 gyrA96 relA1 lac F′[proA⁺B⁺ lacI^(q)ZΔM15::Tn10]        (Tet^(R))),    -   IJ1126, IJ1127, INV110, JM83,    -   JM101 (F′ traD36 proA⁺B⁺ lacI^(q) Δ(lacZ)M15/Δ(lac-proAB) glnV        thi),    -   JM103, JM105, JM106, JM107, JM108,    -   JM109 (F′ traD36 proA⁺B⁺ lacI^(q) Δ(lacZ)M15/Δ(lac-proAB) glnV44        e14⁻ gyrA96 recA1 relA1 endA1 thi hsdR17),    -   JM109(DE3), JM110, JS5, KS1000 (F′ lacI^(q) lac⁺ pro⁺/ara        Δ(lac-pro) Δ(tsp)=Δ(prc)::Kan^(R) eda51::Tn10(Tet^(R))        gyrA(Nal^(R)) rpoB thi-1 argE(am)), LE392,    -   Lemo21(DE3) (fhuA2 [lon] ompT gal (λ DE3) [dcm]        ΔhsdS/pLemo(Cam^(R)) λ DE3=λ sBamHIo ΔEcoRI-B        int::(lacI::PlacUV5::T7 gene1) i21 Δnin5    -   pLemo=pACYC184-PrhaBAD-lysY),    -   LIBRARY EFFICIENCY® DH5A™ (F−φ80lacZΔM15 Δ(lacZYA-argF)U169        recA1 endA1 hsdR17(r_(k) ⁻, m_(k) ⁺) phoA supE44 thi-1 gyrA96        relA1λ−),    -   MACH1™ T1R (F− Φ80lacZΔM15 ΔlacX74 hsdR(rK−, mK+) ΔrecA1398        endA1 tonA),    -   MAX EFFICIENCY® DH10B™ (F-mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15        ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ-rpsL        nupG/pMON14272/pMON7124),    -   MC1061, MC4100, MDS™ 42(MGJ655 fhuACDB(del) endA(del)+deletion        of 699 additional genes, including all IS elements and cryptic        prophages as listed in Posfai et al. (2006) Science        (312):1044-1046), MFDpir,    -   NEB Express l^(q1)(MiniF lacI^(q) (Cam^(R))/fhuA2 [lon] ompT gal        sulA11 R(mcr-73::miniTn10-Tet^(S))2 [dcm]        R(zgb-210::Tn10-Tet^(S)) endA1 Δ(mcrC-mrr)114::IS10),    -   NEB Express, dam⁻/dcm⁻,    -   NEB 5-alpha (fhuA2 Δ(argF-lacZ)U69 phoA glnV44 Φ80Δ (lacZ)M15        gyrA96 recA1 relA1 endA thi-1 hsdR17),    -   NEB 10-beta (Δ(ara-leu) 7697 araD139 fhuA ΔlacX74 galK16 galE15        e14-φ80dlacZΔM15 recA1 relA1 endA1 nupG rpsL (Str^(R)) rph spoT1        Δ(mrr-hsdRMS-mcrBC)),    -   NiCo21(DE3) (can::CBD fhuA2 [lon] ompT gal (λ DE3) [dcm]        arnA::CBD slyD::CBD glmS6Ala ΔhsdS λ DE3=λ sBamHIo ΔEcoRI-B        int::(lacI::PlacUV5::T7 gene1) i21 Δnin5),    -   NM522 (F′ proA⁺B⁺ lacI^(q) Δ(lacZ)M15/Δ(lac-proAB) glnV thi-1        Δ(hsdS-mcrB)5),    -   NOVABLUE™ (endA1 hsdR17 (r_(K12) ⁻ m_(K12) ⁺) supE44 thi-1 recA1        gyrA96 relA1 lac F′[proA⁺B⁺ lacI^(q)ZΔM15::Tn10](Tet^(R))),    -   NovaF− (F⁻ endA1 hsdR17 (r_(K12) ⁻ m_(K12) ⁺) supE44 thi-1 recA1        gyrA96 relA1 lac),    -   NOVAXGF′ ZAPPERS™ (mcrA Δ(mcrC mrr) endA1recA1 φ80dlacZΔM15        ΔlacX74araD139 Δ(ara-leu)7697 galUgalKrpsLnupGλ⁻tonA        F′[lacI^(q)Tn10] (Tet^(R))).    -   OMNIMAX™2T1® (F′ {proAB+ lacIq lacZΔM15 Tn10(TetR) Δ(ccdAB)}        mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 Δ(lacZYA-argF)    -   U169 endA1 recA1 supE44 thi-1 gyrA96 relA1 tonA panD),    -   ONE SHOT® BL21 STAR™ (DE3) (F−ompT hsdSB (rB−, mB−) galdcmrne131        (DE3)),    -   ONESHOT® TOP10 (F− mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 Δ lacX74        recA1 araD139 Δ(araleu)7697galU galK rpsL (StrR) endA1 nupG).    -   ORIGAMI™ (Δ(ara-leu) 7697 ΔlacX74 ΔphoA PvuII phoR araD139 ahpC        galE galK rpsLF′[lac⁺ lacI^(q) pro] (DE3)gor522::Tn10 trxB        (Kan^(R), Str^(R), Tet^(R))),    -   ORAGAMI™ 2 (Δ(ara-leu) 7697 ΔlacX74 ΔphoA PvuII phoR araD139        ahpC galE galK rpsL F′[lac⁺ lacI^(q) pro] gor522::Tn10 trxB        (Str^(R), Tet^(R))),    -   OVEREXPRESS™ C41(DE3) (F− ompT hsdSB (rB− mB−) gal dcm (DE3)),    -   OVEREXPRESS™ C41(DE3)PLYSS (F− ompT hsdSB (rB− mB−) gal dcm        (DE3) pLysS (Cm^(R))),    -   OVEREXPRESS™ C43(DE3) (F− ompT hsdSB (rB− mB−) gal dcm (DE3)),    -   OVEREXPRESS™ C43(DE3)PLYSS (F− ompT hsdSB (rB− mB−) gal dcm        (DE3) pLysS (Cm^(R))),    -   POP2136/pFOS1 (F⁻ glnV44 hsdR17 endA1 thi-1 aroB mal⁻ cI857        lambdaPR),    -   PR1031 (F⁻ thr:Tn10(Tet^(R)) dnaJ259 leu fhuA2 lacZ90(oc) lacY        glnV44 thi),    -   ROSETTA™ (F⁻ompT hsdS_(B)(r_(B) ⁻ m_(B) ⁻) gal dcm pRARE        (Cam^(R))),    -   ROSETTA™ (DE3)PLYSS (F⁻ompT hsdS_(B)(r_(B) ⁻ m_(B) ⁻) gal dcm        (DE3) pLysSRARE2 (Cam^(R))),    -   ROSETTA-GAMI™ (Δ(ara-leu)7697 ΔlacX74 ΔphoA PvuII phoR araD139        ahpC galE galK rpsL F′[lac⁺ lacI^(q) pro] gor522::Tn10 trxB        pRARE2 (Cam^(R), Str^(R), Tet^(R))),    -   ROSETTA-GAMI™ (DE3)PLYSS (Δ(ara-leu)7697 ΔlacX74 ΔphoA PvuII        phoR araD139 ahpC galE galK rpsL (DE3) F′[lac⁺ lacI^(q)        pro]gor522::Tn10 trxB pLysSRARE2 (Cam^(R), Str^(R), Tet^(R))),    -   RR1, RV308, SCARABXPRESS® T7LAC (MDS™42 multiple-deletion        strain (1) with a chromosomal copy of the T7 RNA Polymerase        gene),    -   SS320 (F′[proAB+lacIqlacZΔM15 Tn10 (tet^(r))]hsdR mcrB araD139        Δ(araABC-leu)7679 ΔlacX74 galUgalK rpsL thi),    -   SHUFFLE® (F′ lac pro lacI^(q)/Δ(ara-leu)7697 araD13 fhuA2        Δ(lac)X74 Δ(phoA)PvuII phoR ahpC*galE (or U) galK        Δλatt::pNEB3-r1-cDsbC (SpecR, lacI^(q)) ΔtrxB rpsL150(StrR) Δgor        Δ(malF)3),    -   SHUFFLE® T7 (F′ lac, pro, lacI^(q)/Δ(ara-leu)7697 araD139 fhuA2        lacZ::T7 gene1 Δ(phoA)PvuII phoR ahpC*galE (or U) galK        λatt::pNEB3-r1-cDsbC (Spec^(R), lacI^(q)) ΔtrxB rpsL150(Str^(R))        Δgor Δ(malF)3),    -   SHUFFLE® T7 EXPRESS (huA2 lacZ::T7 gene1 [lon] ompT ahpC gal        λatt::pNEB3-r1-cDsbC (Spec^(R), lacI^(q)) ΔtrxB sulA11        R(mcr-73::miniTn10-Tet^(S))2 [dcm] R(zgb-210::Tn10-Tet^(S))        endA1 Δgor Δ(mcrC-mrr)114::IS10),    -   SOLR (e14-(McrA−) Δ(mcrCB-hsdSMR-mrr)171 sbcC recB recJ uvrC        umuC::Tn5 (Kan^(r)) lac gyrA96 relA1 thi-1 endA1λ^(R) [F′ proAB        lacI^(q)Z ΔM15]^(C) Su−),    -   SCS110, STBL2™ (F− endA1 gln V44 thi-1 recA1 gyrA96 relA1        Δ(lac-proAB) mcrA Δ(mcrBC-hsdRMS-mrr) λ⁻),    -   STBL3™ (F− glnV44 recA13 mcrB mrr hsdS20(rB−, mB−) ara-14 galK2        lacY1 proA2 rpsL20 xyl-5 leu mtl-1),    -   STBL4™ (endA1 glnV44 thi-1 recA1 gyrA96 relA1 Δ(lac-proAB) mcrA        Δ(mcrBC-hsdRMS-mrr) λ⁻ gal F′[proAB⁺ lacI^(q) lacZΔM15 Tn10]),    -   STELLAR™ (F−, endA1, supE44, thi-1, recA1, relA1, gyrA496, phoA,        Φ80d lacZΔ M15, Δ (lacZYA-argF) U169, Δ (mrr-hsdRMS-mcrBC),        ΔmcrA, λ−),    -   SURE (endA1 glnV44 thi-1 gyrA96 relA1 lac recB recJ sbcC        umuC::Tn5 uvrC e14-Δ(mcrCB-hsdSMR-mrr)171 F′[proAB⁺ lacI^(q)        lacZΔM15 Tn10]),    -   SURE2 (endA1 glnV44 thi-1 gyrA96 relA1 lac recB recJ sbcC        umuC::Tn5 uvrC e14-Δ(mcrCB-hsdSMR-mrr) 171 F′[proAB⁺ lacI^(q)        lacZΔM15 Tn10 Amy Cm^(R)]),    -   T7 Express Crystal (fhuA2 lacZ::T7 gene1 [lon] ompT gal sulA11        R(mcr-73::miniTn10-Tet^(S))2 [dcm] R(zgb-210::Tn10-Tet^(S))        endA1 metB1 Δ(mcrC-mrr)114::IS10),    -   T7 Express lysY/I^(q) (MiniF lvsY lacI^(q)(Cam^(R))/fhuA2        lacZ::T7 gene1 [lon] ompT gal sulA11        R(mcr-73::miniTn10-Tet^(S))2 [dcm] R(zgb-210::Tn10-Tet^(S))        endA1 Δ(mcrC-mrr) 114::IS10),    -   T7 Express lysY (MiniF lysY (Cam^(R))/fhuA2 lacZ::T7 gene1 [lon]        ompT gal sulA11 R(mcr-73::miniTn10-Tet^(S))2 [dcm]        R(zgb-210::Tn10-Tet^(S)) endA1 Δ(mcrC-mrr)114::IS10),    -   T7 Express I^(q) (MiniF lacI^(q)(Cam^(R))/fhuA2 lacZ::T7 gene1        [lon] ompT gal sulA11 R(mcr-73::miniTn10-Tet^(S))2 [dcm]        R(zgb-210::Tn10-Tet^(S)) endA1 Δ(mcrC-mrr)114::IS10).    -   T7 Express (fhuA2 lacZ::T7 gene1 [lon] ompT gal sulA11        R(mcr-73::miniTn10-Tet^(S))2 [dcm] R(zgb-210::Tn10-Tet^(S))        endA1 Δ(mcrC-mrr)114::IS10).    -   TB1 (F⁻ ara Δ(lac-proAB) [Φ80dlac Δ(lacZ)M15] rpsL(Str^(R)) thi        hsdR),    -   TG1 (F′ [traD36 proAB⁺ lacI^(q) lacZΔM15]supE thi-1 Δ(lac-proAB)        Δ(mcrB-hsdSM)5, (r_(K) ⁻m_(K) ⁻)),    -   THUNDERBOLT™ GC10 (F− mcrA Δ (mrr-hsdRMSmcrBC) Φ80dlacZ Δ M15        DlacX74 endA1recA1 Δ (ara, leu)7697 araD139 galU galK nupG rpsL        l λ-T1R),    -   UT5600 (F⁻ ara-14 leuB6 secA6 lacY1 proC14 tsx-67d(ompT-fepC)266        entA403 trpE38 rfbD1 rpsL109 xyl-5 mtl-1 thi-1),    -   VEGGIE™ BL21(DE3) (F⁻ompT hsdS_(B)(r_(B) ⁻ m_(B) ⁻) gal        dcm(DE3)), W3110 (λ857S7),    -   WM3064,    -   XL1-Blue (endA1 gyrA96(nal^(R)) thi-1 recA1 relA1 lac glnV44        F′[::Tn10 proAB⁺ lacI^(q)Δ(lacZ)laM15] hsdR17(r_(K) ⁻ r_(K) ⁺)),    -   XL1-Blue MRF′(Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44        thi-1 recA1 gyrA96 relA1 lac [F′ proAB lacI^(q)ZΔM15 Tn10        (Tet^(r))]),    -   XL2-Blue (endA1 gyrA96(nal^(R)) thi-1 recA1 relA1 lac glnV44        F′[::Tn10 proAB⁺ lacI^(q)Δ(lacZ)M15 Amy Cm^(R)] hsdR17(r_(K) ⁻        m_(K) ⁺)).    -   XL2-Blue MRF′(endA1 gyrA96(nal^(R)) thi-1 recA1 relA1 lac glnV44        e14− Δ(mcrCB-hsdSMR-mrr)171 recB recJ sbcC umuC::Tn5 uvrC        F′[::Tn10    -   proAB⁺ lacI^(q)Δ(lacZ)M15 Amy Cm^(R)]),    -   XL1-Red (F− endA1 gyrA96(nal^(R)) thi-1 relA1 lac glnV44        hsdR17(r_(K) ⁻ m_(K) ⁺) mutS mutT mutD5 Tn10),    -   XL10-Gold (endA1 glnV44 recA1 thi-1 gyrA96 relA1 lac Hte        Δ(mcrA)183 Δ(mcrCB-hsdSMIR-mrr)173 tet^(R) F′[proAB        lacI^(q)ZΔM15 Tn10(Tet^(R) Amy Cm^(R))]), and    -   XL10-Gold KanR (endA1 glnV44 recA1 thi-1 gyrA96 relA1 lac Hte        Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr) 173 tet^(R) F′[proAB        lacI^(q)ZΔM15 Tn10(Tet^(R) Amy Tn5(Kan^(R))]).        5. The E. coli host cell of any one of 1-4, wherein the number        of said combinations of said genes either for Table 8 alone,        Table 9 alone, Tables 8 and 9 together, Table 14, or for Tables        8, 9, and 14 together is determined by combination Equation 6:

$\begin{matrix}\frac{n!}{{r!}{\left( {n - r} \right)!}} & {{Equation}\mspace{14mu} 6}\end{matrix}$

-   -   wherein n is the set of genes out of which selection occurs, and    -   r is the number of genes selected for deletion, modification,        and/or inhibition.        6. The E. coli host cell of any one of 1-5, wherein said        combinations of said genes are selected from the group        consisting of:        LTSB01 (genotype: ΔhldD);        LTSB02 (genotype: ΔhldDΔusg);        LTSB03 (genotype: ΔhldDΔusgΔrraA);        LTSB04 (genotype: ΔhldDΔusgΔrraAΔcutA);        LTSB05 (genotype: ΔhldDΔusgΔrraAΔcutAΔnagD);        LTSB06 (genotype: ΔhldDΔusgΔrraAΔcutAΔnagDΔspeA);        LTSB07 (genotype: ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldA);        LTSB08 (genotype: ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnA);        LTSB09 (genotype: ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetE);        LTSB010 (genotype:        ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgt);        LTSB011 (genotype:        ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargG);        LTSB012 (genotype:        ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypA);        LTSB013 (genotype:        ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentF);        LTSB014 (genotype:        ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaO);        LTSB015 (genotype:        ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyD);        LTSB016 (genotype:        ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgatZ);        LTSB017 (genotype:        ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgatZΔilvB);        LTSB018 (genotype:        ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgatZΔilvBΔ        glgP);        LTSB019 (genotype:        ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgatZΔilvBΔglgPΔnusA);        and        LTSB020 (genotype:        ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgatZΔilvBΔglgPΔnusAΔmetH).

In any of the gene combinations in 6 comprising multiple genes, one ormore of these genes can be omitted as long as the resulting E. coli hostcells exhibit growth rates and/or viability and/or capacity forexpression of target molecules in the range of from about 60% to about100%, or more; or from about 70% to about 100%, or more; or from about75% to about 100%, or more, of that of the parent cells from which theyare derived, and as long as chromatographic separation capacity isimproved in an amount in the range of from about 5% to about 35%, ormore; from about 5% to about 40%, or more; from about 5% to about 45%,or more; from about 5% to about 50%; or more, and so on similarly,compared to that of parent cells from which they are derived, dependingon the number and particular combination of genes deleted, modified, orinhibited.

In addition to these various gene omissions, these E. coli host cellscan also further comprise (or combine in addition to these omissions)deletion, modification, and/or inhibition/reduction of expression of oneor more essential genes selected from among rpoB, rpoC, tufa, ycfD,groL, prs, fusA, hemL, slyD, infB, mukB, and rnt as long as theresulting E. coli host cells exhibit growth rates and/or viabilityand/or capacity for expression of target molecules in the range of fromabout 60% to about 100%, or more; or from about 70% to about 100%, ormore; or from about 75% to about 100%, or more, of that of the parentcells from which they are derived, and as long as chromatographicseparation capacity is improved in an amount in the range of from about5% to about 35%, or more; from about 5% to about 40%, or more; fromabout 5% to about 45%, or more; from about 5% to about 50%; or more, andso on similarly, compared to that of parent cells from which they arederived, depending on the number and particular combination of genesmodified or inhibited. Modification of these essential genes can beperformed, for example, by the methods described below in thedescription of this disclosure, and/or could be circumvented by thefeeding strategies also discussed below, depending on which essentialgene(s) is(are) deleted, modified, and/or the expression of which isinhibited or reduced.

7. The E. coli host cell of any one of 1-6, wherein said chromatographicseparation efficiency of said target host cell or target recombinantpeptide, polypeptide, or protein is improved compared to thechromatographic separation efficiency of said target host cell or targetrecombinant peptide, polypeptide, or protein in the presence ofpeptides, polypeptides, or proteins coded for by said genes that aredeleted, modified, and/or the expression of which is reduced orcompletely inhibited in said E. coli host cell upon affinity oradsorption, non-affinity column chromatography of said target host cellor target recombinant peptide, polypeptide, or protein.8. The E. coli host cell of 7, wherein improvement of saidchromatographic separation efficiency of said target host cell or targetrecombinant peptide, polypeptide, or protein is in the range of fromabout from about 5% to about 35%, or more; from about 5% to about 40%,or more; from about 5% to about 45%, or more; from about 5% to about50%; or more, or from about 10% to about 20%, compared tochromatographic separation efficiency of said target host cell or targetrecombinant peptide, polypeptide, or protein in the presence ofpeptides, polypeptides, or proteins coded for by said genes that aredeleted, modified, and/or the expression of which is reduced orcompletely inhibited in said E. coli host cell upon affinity oradsorption, non-affinity column chromatography of said target host cellor target recombinant peptide, polypeptide, or protein.9. The E. coli host cell of any one of 1-8, wherein said chromatographicseparation efficiency is independent of elution conditions under whichsaid target host cell or target recombinant peptide, polypeptide, orprotein emerges from an affinity or adsorption, non-affinitychromatography column as an enriched fraction.10. The E. coli host cell of any one of 1-9, wherein deletion of saidgenes is performed by homologous recombination or frame shift mutation.11. The E. coli host cell of any one of 1-9, wherein modification ofsaid genes is performed by a method selected from the group consistingof point mutation; isozyme substitution; transposon mutagenesis;RNA-guided nucleases employing CRISPR-cas technology; and replacement bya gene from another organism that performs the same or similar functionand that does not significantly adversely affect chromatographicseparation efficiency and separation capacity, or growth, viability, orcapacity for expression of said host cell, selected from amongheterologs, homologs, analogs, paralogs, orthologs, and xenologs.12. The E. coli host cell of any one of 1-9, wherein expression of saidgenes is reduced or completely inhibited by a method selected from thegroup consisting of RNA silencing, antisense oligonucleotide inhibition,and replacement of a native promoter with a weaker promoter.13. The E. coli host cell of any one of 1-12, which exhibits about 5%,or more, to about 100%, or more; or from about 60% to about 100%, ormore; or from about 70% to about 100%, or more; or from about 75% toabout 100%, or more of the viability, growth rate, or capacity forexpression of said target host cell or target recombinant peptide,polypeptide, or protein expressed in said E. coli host cell compared tothat of said parent cell from which it is derived, or which exhibitsviability, growth rate, or capacity for expression of said target hostcell or target recombinant peptide, polypeptide, or protein expressed insaid E. coli host cell greater than that of said parent cell from whichit is derived.14. The E. coli host cell of any one of 1-13, wherein said target hostcell or target recombinant peptide, polypeptide, or protein is presentin a lysate of said E. coli host cell, or is secreted by said E. colihost cell.15. The E. coli host cell of any one of 1-14, wherein said target hostcell or target recombinant peptide, polypeptide, or protein is anendogenous peptide, polypeptide, or protein.16. The E. coli host cell of 15, wherein said endogenous peptide,polypeptide, or protein is selected from the group consisting of anuclease, a ligase, a polymerase, an RNA- or DNA-modifying enzyme, acarbohydrate-modifying enzyme, an isomerase, a proteolytic enzyme, and alipolytic enzyme.17. The E. coli host cell of any one of 1-14, wherein said targetrecombinant peptide, polypeptide, or protein is a heterologous peptide,polypeptide, or protein.18. The E. coli host cell of 17, wherein said heterologous peptide,polypeptide, or protein is selected from the group consisting of anenzyme and a therapeutic peptide, polypeptide, or protein.19. The E. coli host cell of 18, wherein said enzyme is selected fromthe group consisting of a nuclease, a ligase, a polymerase, an RNA- orDNA-modifying enzyme, a carbohydrate-modifying enzyme, an isomerase, aproteolytic enzyme, and a lipolytic enzyme, and said therapeuticpeptide, polypeptide, or protein is selected from the group consistingof antibody, an antibody fragment, a vaccine, an enzyme, a growthfactor, a blood clotting factor, a hormone, a nerve factor, aninterferon, an interleukin, tissue plasminogen activator, and insulin.20. The E. coli host cell of any one of 1-19, wherein said reducedgenome compared to the genome in the parent cell from which it isderived is less than 5% smaller, less than about 4.5% smaller, less thanabout 4% smaller, less than about 3.5% smaller, less than about 3%smaller, less than about 2.5% smaller, less than about 2% smaller, lessthan about 1.5% smaller, or less than about 1% smaller, than the genomeof said parent cell from which it is derived.21. The E. coli host cell of any one of 1-19, wherein said reducedgenome compared to the genome in the parent cell from which it isderived is between about 4.17 Mb to about 4.346 Mb.22. The E. coli host cell of any one of 1-21, wherein genes that aredeleted, modified, and/or the expression of which is reduced orcompletely inhibited in said host cell compared to expression of saidgenes in said parent cell from which it is derived code for proteinsthat impair the chromatographic separation efficiency of said targetrecombinant peptide, polypeptide, or protein expressed in said host cellin the presence of peptides, polypeptides, or proteins coded for by saidgenes that are deleted, modified, and/or the expression of which isreduced or completely inhibited in said host cell, and that elute from achromatographic affinity column having a ligand, in a buffer comprisinga compound that dictates adsorption to its respective ligand duringequilibration and elution from said affinity column, in an amount in therange, in a combination selected from the group consisting of thecombinations in the following table:

Compound in Buffer That Dictates Adsorption to Affinity Column DuringEquilibration and Causes Ligand Elution From Column Concentration or pHRange Glutathione Glutathione from about 0 mM to about 10 mMS-transferase Amino acid A common salt from about 0 mM to about 2M(e.g., lysine) Amino acid pH from about pH 2 to about pH 11 Avidin Achaotropic salt from about 0M to about 4M Avidin pH from about pH 2 toabout pH 10.5 Carbohydrate Sugar or isocratic from about 0 mM to about10 mM (e.g., Dextrin) (e.g., maltose) Carbohydrate pH from about pH 5 toabout pH 8 Organic dye A common salt from about 0 mM to about 1.5M(e.g., Cibacron Blue) Organic dye pH from about pH 4 to about pH 8Organic dye Imidazole from about 5 mM to about 250 mM or a common saltDivalent metal pH from about pH 4 to about pH 12 (e.g., Ni(II)) Divalentmetal Imidazole from about 5 mM to about 500 mM (e.g., Ni(II)) Heparin Acommon salt from about 0 mM to about 2M Protein A or Protein G Glycinefrom about 0 mM to about 100 mM Protein A or Protein G pH from about pH3 to about pH 7 IgG Glycine from about 0 mM to about 100 mM CoenzymeCompeting Protein from about 1 mM to about 12 mM23. The E. coli host cell of any one of 1-21, wherein genes that aredeleted, modified, and/or the expression of which is reduced orcompletely inhibited in said host cell compared to expression of saidgenes in said parent cell from which it is derived, code for proteinsthat impair the chromatographic separation efficiency of said targethost cell or target recombinant peptide, polypeptide, or proteinexpressed in said host cell in the presence of peptides, polypeptides,or proteins coded for by said genes that are deleted, modified, and/orthe expression of which is reduced or completely inhibited in said hostcell, and that elute from a chromatographic adsorption, non-affinitycolumn having a ligand, in a buffer comprising a compound that dictatesadsorption to its respective ligand during equilibration and elutionfrom said adsorption, non-affinity column, in an amount in the range, ina combination selected from the group consisting of the combinations inthe following table:

Compound in Buffer That Dictates Adsorption to Non-Affinity ColumnDuring Equilibration and Causes Elution From Ligand Column Concentrationor pH Range Ion exchange Common salt from about 0M to about 2M Ionexchange pH from about pH 2 to about pH 12 Reverse phase Organic solventfrom about 0% to about 100% Hydrophobic Common salt from about 2M toabout 0M interaction24. The E. coli host cell of 22 or 23, wherein said common salt isselected from the group consisting of a chloride salt, a sulfate salt,an acetate salt, a carbonate salt, and a propionate salt.25. The E. coli host cell of 23, wherein said organic solvent isselected from the group consisting of acetonitrile, methanol, and2-propanol.26. The E. coli host cell of any one of 1-25, wherein increasedseparation efficiency is manifested as increased separation capacity,increased separation selectivity, or both.27. The E. coli host cell of 26, wherein separation capacity is definedas:

-   -   i) the amount of target host cell or target recombinant peptide,        polypeptide, or protein adsorbed to said column per mass lysate        in the case where said target host cell or target recombinant        peptide, polypeptide, or protein is not secreted, or mass        culture medium in the case where said target host cell or target        recombinant peptide, polypeptide, or protein is secreted,        applied to said column, and separation selectivity is defined as        the amount of target host cell or target recombinant peptide,        polypeptide, or protein adsorbed to said column per total        peptide, polypeptide, or protein adsorbed to said column, or    -   ii) the amount of host cell peptides, polypeptides, or proteins        adsorbed by a column per mass lysate fed to the column in the        absence of expression of a target recombinant peptide,        polypeptide, or protein.        28. The E. coli host cell of 26 or 27, wherein said increased        separation capacity is in the range of from about 5% to about        35%, or more; from about 5% to about 40%, or more; from about 5%        to about 45%, or more; or from about 5% to about 50%, or more.        29. The E. coli host cell of any one of 1-28, wherein separation        of said target host cell or target recombinant peptide,        polypeptide, or protein from host cell peptides, polypeptides,        or proteins is performed by column chromatography employing a        solid phase chromatography medium.        30. The E. coli host cell of 29, wherein said column        chromatography is selected from the group consisting of affinity        chromatography employing an affinity ligand bound to said solid        phase, and adsorption-based, non-affinity chromatography.        31. The host cell of 30, wherein said affinity ligand is        selected from the group consisting of an amino acid, a divalent        metal ion, a carbohydrate, an organic dye, a coenzyme,        glutathione S-transferase, avidin, heparin, protein A, and        protein G.        32. The host cell of 31, wherein said divalent metal ion is        selected from the group consisting of Cu⁺⁺, Ni⁺⁺, Co⁺⁺, and        Zn⁺⁺; said carbohydrate is selected from the group consisting of        maltose, arabinose, and glucose; said organic dye is a dye        comprising a triazene moiety; and said coenzyme is selected from        the group consisting of NADH and ATP.        33. The E. coli host cell of 30, wherein said adsorption-based,        non-affinity chromatography is selected from the group        consisting of ion exchange chromatography, reverse phase        chromatography, and hydrophobic interaction chromatography.        34. The E. coli host cell of 33, wherein said adsorption-based,        non-affinity chromatography is ion exchange chromatography.        35. The E. coli host cell of 34, wherein said ion exchange        chromatography employs a ligand selected from the group        consisting of diethylaminoethyl cellulose (DEAE), monoQ or other        Q resin, and S.        36. The E. coli host cell of any one of 1-35, wherein said host        cell peptides, polypeptides, or proteins that impair separation        efficiency of said target host cell or target recombinant        peptide, polypeptide, or protein expressed in said host cell are        peptides, polypeptides, or proteins that are strongly retained        during column chromatography.        37. The E. coli host cell of 36, wherein said host cell        peptides, polypeptides, or proteins that are strongly retained        during ion exchange chromatography are those that are retained        during elution with a mobile phase comprising a common salt in        the range of from about 5 mM to about 2,000 mM.        38. The E. coli host cell of 36, wherein said host cell        peptides, polypeptides, or proteins that are strongly retained        during ion exchange chromatography are those that are retained        during elution with a mobile phase comprising a common salt in        the range of from about 500 mM to about 1,000 mM.        39. The E. coli host cell of any one of 1-35, wherein said host        cell peptides, polypeptides, or proteins that impair the        separation efficiency of said target host cell or target        recombinant peptide, polypeptide, or protein expressed in said        host cell are peptides, polypeptides, or proteins that are        weakly retained during column chromatography.        40. The E. coli host cell of 39, wherein said host cell        peptides, polypeptides, or proteins that are weakly retained        during chromatography are those that are retained during elution        with a mobile phase comprising a common salt in the range of        from about 5 mM to about 500 mM.        41. The E. coli host cell of 39, wherein said host cell        peptides, polypeptides, or proteins that are weakly retained        during chromatography are those that are retained during elution        with a mobile phase comprising a common salt in the range of        from about 10 mM to about 350 mM.        42. The E. coli host cell of any one of 1-35, wherein said host        cell peptides, polypeptides, or proteins that impair the        separation efficiency of said target host cell or target        recombinant peptide, polypeptide, or protein expressed in said        host cell are peptides, polypeptides, or proteins that are both        strongly retained and weakly retained during column        chromatography.        43. A method of enriching the amount of a target host cell or        target recombinant peptide, polypeptide, or protein relative to        other peptides, polypeptides, or proteins present in an initial        protein mixture comprising said target host cell or target        recombinant peptide, polypeptide, or protein, comprising the        steps of:    -   i) selecting a chromatography medium that binds said target host        cell or target recombinant peptide, polypeptide, or protein from        the group consisting of an affinity chromatography medium and an        adsorption-based, non-affinity chromatography medium;    -   ii) in the case where an affinity chromatography medium is        selected, expressing said target host cell or target recombinant        peptide, polypeptide, or protein in said E. coli host cell of        any one of 1-22, 24, 26-32, or 36-42;    -   iii) in the case where an adsorption-based, non-affinity        chromatography medium is selected, expressing said target host        cell or recombinant peptide, polypeptide, or protein in said E.        coli host cell of any one of 1-21, 23-30, or 33-42; and    -   iv) chromatographing said initial protein mixture comprising        said target host cell or target recombinant peptide,        polypeptide, or protein using said chromatography medium of        step i) or step ii), as appropriate, and collecting elution        fractions, thereby obtaining one or more fractions containing an        enriched amount of said target host cell or target recombinant        peptide, polypeptide, or protein relative to other peptides,        polypeptides, or proteins in said fraction compared to the        amount of said target host cell or target recombinant peptide,        polypeptide, or protein relative to other peptides,        polypeptides, or proteins in said initial protein mixture.        44. The method of 43, further comprising chromatographing an        enriched fraction of step iv) to obtain said target host cell or        target recombinant peptide, polypeptide, or protein in a desired        degree of purity.        45. The method of 44, further comprising recovering said target        host cell or target recombinant peptide, polypeptide, or        protein.        46. A method of preparing a pharmaceutical or veterinary        composition comprising a therapeutic peptide, polypeptide, or        protein, comprising the steps of:    -   i) selecting a chromatography medium that binds said therapeutic        peptide, polypeptide, or protein from the group consisting of an        affinity chromatography medium and an adsorption-based,        non-affinity chromatography medium;    -   ii) in the case where an affinity chromatography medium is        selected, expressing said therapeutic peptide, polypeptide, or        protein in said E. coli host cell of any one of 1-22, 24, 26-32,        or 36-42;    -   iii) in the case where an adsorption-based, non-affinity        chromatography medium is selected, expressing said therapeutic        peptide, polypeptide, or protein in said E. coli host cell of        any one of 1-21, 23-30, or 33-42; and    -   iv) in the case where said therapeutic peptide, polypeptide, or        protein is not secreted from said E. coli host cell, preparing a        lysate of said host cell containing said therapeutic peptide,        polypeptide, or protein, producing an initial therapeutic        peptide-, polypeptide-, or protein-containing mixture; or    -   v) in the case where said therapeutic peptide, polypeptide, or        protein is secreted from said E. coli host cell, harvesting        culture medium in which said host cell is grown, containing said        therapeutic peptide, polypeptide, or protein, thereby obtaining        an initial therapeutic peptide-, polypeptide-, or        protein-containing mixture;    -   vi) chromatographing said initial therapeutic peptide-,        polypeptide-, or protein-containing mixture of step iv) or        step v) using said chromatography medium of step i) or step ii),        as appropriate, and collecting elution fractions, thereby        obtaining one or more fractions containing an enriched amount of        said therapeutic peptide, polypeptide, or protein relative to        other peptides, polypeptides, or proteins in said fraction        compared to the amount of said therapeutic peptide, polypeptide,        or protein relative to other peptides, polypeptides, or proteins        in said initial protein mixture;    -   vii) optionally, further chromatographing an enriched fraction        of step vi) to obtain said therapeutic peptide, polypeptide, or        protein in a desired degree of purity; and    -   viii) recovering said therapeutic peptide, polypeptide, or        protein.

Step vii) is optional, depending on the intended use of the therapeuticand regulatory agency requirements for that use, as not all treatmentregimens in which therapeutics are employed require high degrees ofpurity, i.e., less pure preparations may suffice.

47. The method of 46, further comprising formulating said therapeuticpeptide, polypeptide, or protein with a pharmaceutically or veterinarilyacceptable carrier, diluent, or excipient to produce a pharmaceutical orveterinary composition, respectively.48. The method of 46 or 47, wherein said therapeutic peptide,polypeptide, or protein is produced recombinantly in said E. coli hostcell.49. A method of purifying an enzyme, comprising the steps of:

-   -   i) selecting a chromatography medium that binds said enzyme from        the group consisting of an affinity chromatography medium and an        adsorption-based, non-affinity chromatography medium;    -   ii) in the case where an affinity chromatography medium is        selected, expressing said enzyme in said E. coli host cell of        any one of 1-22, 24, 26-32, or 36-42;    -   iii) in the case where an adsorption-based, non-affinity        chromatography medium is selected, expressing said enzyme in        said E. coli host cell of any one of 1-21, 23-30, or 33-42;    -   iv) in the case where said enzyme is not secreted from said E.        coli host cell, preparing a lysate of said host cell containing        said enzyme, producing an initial enzyme-containing mixture; or    -   v) in the case where said enzyme is secreted from said E. coli        host cell, harvesting culture medium in which said host cell is        grown, containing said enzyme, thereby obtaining an initial        enzyme-containing mixture;    -   vi) chromatographing said initial enzyme-containing mixture of        step iv) or step v) using said chromatographic medium of step i)        or step ii), as appropriate, and collecting elution fractions,        thereby obtaining one or more fractions containing an enriched        amount of said enzyme relative to other peptides, polypeptides,        or proteins in said fraction compared to the amount of said        enzyme relative to other peptides, polypeptides, or proteins in        said initial protein mixture;    -   vii) optionally, further chromatographing an enriched fraction        of step vi) to obtain said enzyme in a desired degree of purity;        and    -   viii) recovering purified enzyme.

Step vii) is optional, depending on the intended use of the enzyme, asnot all processes in which enzymes are employed require high degrees ofpurity, i.e., “crude” enzyme preparations may suffice.

50. The method of 49, further comprising placing said purified enzyme ina buffer solution in which said purified enzyme is stable and retainsenzymatic activity.51. The method of 50, wherein said purified enzyme-containing buffersolution is reduced to dryness.52. The method of 51, wherein said dry purified enzyme-containing buffersolution is in the form of a powder.53. The method of any one of 49-52, wherein said enzyme is producedrecombinantly in said E. coli host cell.54. A kit, comprising said E. coli host cell of any one of 1-42.55. The kit of 54, further comprising instructions for expressing atarget host cell or target recombinant peptide, polypeptide, or proteinin said E. coli host cell.56. The kit of 55, wherein said target recombinant peptide, polypeptide,or protein is an endogenous or heterologous peptide, polypeptide, orprotein.57. The kit of any one of 54-56, wherein said instructions furthercomprise directions for purifying said expressed target host cell orrecombinant peptide, polypeptide, or protein by affinity chromatographyor adsorption-based, non-affinity chromatography.58. The kit of any one of 54-57, further comprising a chromatographicresin for affinity chromatography or adsorption-based, non-affinitychromatography.59. A method of enriching a target peptide, polypeptide, or protein froma mixture of peptides, polypeptides, or proteins obtained from an E.coli host cell of any one of 1-42, comprising the steps of:

-   -   a. chromatographing said mixture via affinity chromatography or        adsorption-based, non-affinity chromatography;    -   b. collecting an elution fraction that contains an enriched        amount of said target peptide, polypeptide, or protein in said        fraction compared to the amount of said target peptide,        polypeptide, or protein in said mixture; and    -   c. recovering said target peptide, polypeptide, or protein from        said elution fraction.

Step c. can be optional.

60. The method of 59, wherein said mixture is a lysate of said E. colihost cell in the case where said target peptide, polypeptide, or proteinaccumulates intracellularly, or is medium in which said E. coli hostcell is grown in the case where said target peptide, polypeptide, orprotein is secreted by said host cell.61. The method of 59 or 60, further comprising chromatographing saidtarget peptide, polypeptide, or protein of step c. in order to obtainsaid target peptide, polypeptide, or protein in a desired degree ofpurity.62. The method of 61, further comprising recovering purified targetpeptide, polypeptide, or protein.63. The method of any one of 59-62, wherein said target peptide,polypeptide, or protein is an endogenous host cell peptide, polypeptide,or protein.64. The method of any one of 59-62, wherein said target peptide,polypeptide, or protein is an endogenous or heterologous peptide,polypeptide, or protein that is recombinantly expressed in said E. colihost cell.

Further scope of the applicability of the present invention will becomeapparent from the detailed description and drawings provided below.However, it should be understood that the detailed description andspecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other aspects, features, and advantages of the presentinvention will be better understood from the following detaileddescriptions taken in conjunction with the accompanying drawing(s), allof which are given by way of illustration only, and are not limitativeof the present invention, in which:

FIG. 1 shows a Circos® rendering of model data used to describe multipleseparatomes. In the figure, the ring is comprised of segments thatrepresent either gene positions or % B. Four different separatomesassociated with popular methods of chromatography (IMAC, immobilizedmetal affinity chromatography; AEX, anion exchange chromatography; CEX,cation exchange chromatography; and HIC, hydrophobic interactionchromatography) are represented. Connecting lines map individualproteins contained within a separatome to their gene (located on theouter ring), with the concentric (inner gray) ring describing theconcentration of the protein found in the fractions as they elute from acolumn as indicated by the length of the black bar segments. Other datathat could be depicted in a Circos® rendering include gene designation,essentiality of gene product, metabolic category, or other parameter,placed on a series of concentric rings or attached to the connectinglines, for example as shown by the other concentric ring fragment.

FIG. 2 shows a Circos® rendering of model data describing the separatomeof E. coli for ion exchange chromatography. Similar to FIG. 1 is the useof connecting lines that indicate genes associated with proteins foundin the separatome of E. coli for a particular resin/equilibratingcondition. However, this rendering provides detail as to the elutionfraction by connecting a gene to a particular box on the ring thatrepresents a salt concentration. The lower black fragment of the circleentitled “Escherichia coli genome” can contain the location of genespresent on the E. coli chromosome. Each box represents a different cutfrom a column.

FIG. 3 shows the distribution of proteins contained within various IMACfractions that elute from a Ni(II) column. In particular, note the lowconcentration of host cell proteins within the 120 mM fraction.

FIG. 4 shows a Western blot (a) and protein gel (b) that indicate lackof expression of gene products of yfbG, adhP, and cyoA. Lack ofexpression is indicated by absence of spot or band.

FIG. 5 shows removal of thyA prior to homologous recombination.

FIG. 6 shows removal of a gene targeted for deletion via a two stepprocess.

FIG. 1 relates to the detailed description of the invention.

FIGS. 2, 5, and 6 relate to Example 2, Construction of the Ion ExchangeSeparatome of E. coli and Its Use to Design and Build Novel Host Strainsfor a Common Chromatography Resin.

FIGS. 3 and 4 refer to Example 1, Identification of Host Cell ProteinsAssociated With a Specific Product, Histidine-Tagged Green FluorescentProtein, as a Comparative Example.

FIG. 7 shows the electrophoresis of the PCR amplification of each targetgene after deletion as described in Example 2, Section II.

FIG. 8 shows the results of fed-batch growth studies in Example 2,Section II.

FIG. 9 shows the results of a fed-batch growth study and a standardbatch growth study in Example 2. Section II.

FIG. 10 shows 207 mM bound fraction for the knockout and control strainas described in Example 2, Section II. As shown, the bands correspondingto the gene products of rnr, tgt, and ycaO disappear in the LTS05+tstrain. The bands corresponding to metH and entF are not visible becausethey do not typically bind at this salt concentration. Known keycontaminants, hldD, ptsI, usg, and rraA are also labeled on the gel.

FIG. 11 shows ECP analysis of LTS05+t as described in Example 2, Section11. The top portion of the figure shows the FPLC chromatogram with theA₂₈₀ on the left axis and % buffer B on the right axis. Below thechromatogram is a table showing the % buffer B converted into a mM saltconcentration followed by the measured protein concentration in thewindow. The third row of the table shows a breakdown of how all of theapplied proteins distributed over the elution windows as a percentagecalculated as mg protein in elution window/total mg of protein appliedto the column. The fourth row focuses on the breakdown of just the boundprotein into specific windows and gives a percentage calculated as mgprotein in the window/mg protein in all bound fractions (59.5 mM to 1000mM). This number provides the best indication of how the ECP changed inthe knockout strain. The bottom of the figure shows the SDS-PAGE gel forthe sample.

FIG. 12 shows ECP analysis of MG1655 as described in Example 2, SectionII. The description of the figure is the same as that for FIG. 11.

FIG. 13 shows that if the percent of bound protein for both the knockoutstrain and MG1655 are added cumulatively, the result is a measure of thecolumn loading profile as described in Example 2, Section II.

FIG. 14 shows the growth rates of parent E. coli strain MG1655 andknockout strain LTSF06 in Example 3.

FIG. 15 shows the amount of proteins in parent E. coli strain MG1655 andknockout strain LTSF06 bound to DEAE at 5 mM NaCl (Example 3).

FIG. 16 shows the amount of proteins in parent E. coli strain MG1655 andknockout strain LTSF06 bound to DEAE at 100 mM NaCl (Example 3).

FIG. 17 shows the amount of proteins in parent E. coli strain MG1655 andknockout strain LTSF06 bound to DEAE at 250 mM NaCl (Example 3).

DETAILED DESCRIPTION

The following detailed description is provided to aid those skilled inthe art in practicing the various embodiments of the present invention.Even so, the following detailed description should not be construed tounduly limit the present invention, as modifications and variations inthe embodiments herein discussed may be made by those of ordinary skillin the art without departing from the spirit or scope of the presentinventive discovery.

The present disclosure is explained in greater detail below. Thisdisclosure is not intended to be a detailed catalog of all the differentways in which embodiments of this disclosure can be implemented, or allthe features that can be added to the instant embodiments. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure, which variations and additions do not depart fromthe scope of the instant disclosure. Hence, the following specificationis intended to illustrate some particular embodiments of the disclosure,and not to exhaustively specify all permutations, combinations, andvariations thereof encompassed by the present invention.

Any feature, or combination of features, described herein is(are)included within the scope of the present disclosure, provided that thefeatures included in any such combination are not mutually inconsistentas will be apparent from the context, this specification, and theknowledge of one of ordinary skill in the art. Additional advantages andaspects of the present disclosure are apparent in the following detaileddescription and claims.

By way of example, and not limitation, calculation and identification ofcombinations of genes useful in the E. coli host cells and methodsdisclosed herein, as mathematically described in Example 2, are equallyapplicable to the list of genes disclosed in Table 14 in Example 3.

The contents of each of the references cited herein, including journalliterature and trade publications, patent applications, patents, etc.,are herein incorporated by reference in their entirety. In case ofconflict, the present specification, including explanations of terms,will control.

As noted above, Asenjo et al. (2004). “Is there a rational method topurify proteins? From expert systems to proteomics”, Journal ofMolecular Recognition 17:236-247, points out that, “Until now, it hasbeen virtually impossible to select separation and purificationoperations for proteins either for therapeutic or analytical applicationin a rational manner due to lack of fundamental knowledge on themolecular properties of the materials to be separated and the lack of anefficient system to organize such information.” The present methods andhost cells provide solutions to this problem.

A principal consideration in the production of recombinant products, bethey therapeutic molecules, enzymes for industrial or diagnosticpurposes, or for other commercial applications, is the time and costinvolved in purification of the biological. Downstream (post cellculture) bottlenecks present as either reduced capacity (mg targetmolecule captured/volume unit operation), low purification efficiency(mg target molecule captured/total mg), or combination thereof, and whenthey are encountered, limit the ability to screen material(s) underdevelopment or to manufacture candidate molecules. As demonstratedherein, the inventors have developed efficient alternatives to affinitychromatography via strategic changes to the E. coli host cell genomethat reduce the burden imposed by the presence of host cell proteins.

The embodiments disclosed herein include a separatome-based proteinexpression and purification platform based on the juxtaposition of thechromatographic binding properties of genomic peptides, polypeptides,and proteins with the characteristic and location of genes on the targetchromosome, such as those of E. coli, Bacillus subtilis, yeasts, andother host cells. The separatome-based protein expression andpurification platform maps the separatome of target chromosomes based onrelationships between the loci of genes associated with nuisancepeptides, polypeptides, and proteins. In addition, the separatome-basedprotein expression and purification platform reduces the genome of hostcells through precisely targeted modifications to create custom, robusttarget host strains with reduced nuisance peptides, polypeptides, andproteins. Moreover, the present separatome-based protein expression andpurification platform provides a computerized knowledge tool that, givenseparatome data regarding a target peptide, polypeptide, or protein,intuitively suggests strategies leading to efficient purification. Theseparatome-based protein expression and purification platform is anefficient bioseparation system that intertwines host cell strain andchromatography.

As disclosed below in Examples 2 and 3, the inventors have identifiedthe genes listed in Tables 8, 9, and 14 as preferred, high prioritycandidates for improving the chromatographic separation efficiency oftarget host cell or target recombinant peptides, polypeptides, orproteins expressed in the E. coli host cells disclosed herein viaaffinity or non-affinity, adsorption chromatography.

As exemplified by the genes listed in descending rank order ofimportance in Tables 8, 9, and 14 in Examples 2 and 3, importance scoreequation 3 identifies highly impactful E. coli parent cell HCPs thatadversely affect chromatographic separation capacity. As high prioritycandidates for deletion, modification, or inhibition to constructimproved E. coli host cell strains for target peptide, polypeptide, andprotein expression and purification, it is highly likely that most, ifnot all, combinations of these genes will be effective in improvingseparation efficiency, including separation capacity, of targetbiomolecules from host cells in which these biomolecules are expressedand in which combinations of these nuisance genes are deleted, modified,and/or inhibited. Preferred gene combinations for deletion, etc., arethose that improve chromatographic separation capacity in the range offrom about 50% to about 35%, or more; from about 5% to about 40%, ormore; from about 5% to about 45%, or more; from about 5% to about 50%,or more, and so on similarly depending on the number and particularcombination of genes deleted, etc., and that still permit cells toexhibit growth rates and/or viability and/or capacity for expression oftarget molecules in the range of from about 60% to about 100%, or more;or from about 70% to about 100%, or more; or from about 75% to about100%, or more, compared to that of the parent cells from which they arederived. The presently disclosed methods and highly ranked genesidentified out of the thousands of genes present in the E. coli genometherefore guide construction of improved E. coli host cells exhibitingimproved separation capacity and satisfactory growth, viability, andcapacity for expression and purification of target peptides,polypeptides, and proteins without the need for hit or miss undueexperimentation within the astronomically large numbers of possible genecombinations within the E. coli genome.

The effectiveness of any of the various possible combinations of highranking genes targeted for deletion, modification, or inhibitionselected from either Table 8 alone, Table 9 alone. Table 14 alone, orany combination of these tables, in improving chromatographic separationefficiency of target host cell or target recombinant peptides,polypeptides, and proteins can easily be determined by the methodsdisclosed herein.

As exemplified in Example 3, collecting and interpreting data on the E.coli proteome facilitates the design and construction of an improvedhost cell strain that when lysed and passed over an anion exchangecolumn displays a significant reduction in adsorption of host cellproteins (ca. 15% depending on conditions). Further development of thisand other strains by the methods disclosed herein will facilitateaffinity chromatography-like efficiencies with common and less expensivechromatographic matrices.

The data presented in Examples 2 and 3 demonstrate that the presentseparatome concept, including importance equation 3, facilitatesreduction in host cell proteins (HCPs) encountered during bioprocessing,improving column capacity and overall chromatographic separationefficiency, without adversely impacting host cell growth, viability, orcapacity for expression, and that this can be achieved in a rational,stepwise predictable manner. Results obtained with the E. coli knockoutstrain LTSF06 in example 3 demonstrate that with strategic deletions,significant improvement in column efficiency can occur. Identificationand ordering of several dozen high ranking genes as determined from theimportance equation disclosed herein out of the thousands of genes inthe E. coli genome facilitates maximum improvements in E. coli hostcells used for expression of a wide range of recombinant productswithout having to engineer individual host cells for specificrecombinant targets. While other investigations have considered knockoutor mutation to improve the purity of a single recombinant product, themathematical framework disclosed herein guides minimal changes that canbe made to the E. coli genome that are useful regardless of targetrecombinant product. These minimal, but strategic, changes positivelyaffect the initial chromatographic capture step, identified as a keybottleneck by biotherapeutic and enzyme manufacturers. Improvedseparation capacity extends the run time of the column over an increasedvolume of the feedstock run through the column, and improves the bindingefficiency/binding selectivity of the target recombinant molecule,improving separation of the target molecule from contaminating host cellproteins.

DEFINITIONS

The following definitions are provided to aid the reader inunderstanding the various aspects of the present invention. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by those of ordinary skill inthe art to which the invention pertains.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art.Similarly, the word “or” is intended to include “and” unless the contextclearly indicates otherwise. Hence “comprising A or B” means includingA, or B, or A and B. Furthermore, the use of the term “including”, aswell as other related forms, such as “includes” and “included”, is notlimiting.

The term “about” as used herein is a flexible word with a meaningsimilar to “approximately” or “nearly”. The term “about” indicates thatexactitude is not claimed, but rather a contemplated variation. Thus, asused herein, the term “about” means within 1 or 2 standard deviationsfrom the specifically recited value, or ±a range of up to 20%, up to15%, up to 10%, up to 5%, or up to 4%, 3%, 2%, or 1% compared to thespecifically recited value.

The term “comprising” as used in a claim herein is open-ended, and meansthat the claim must have all the features specifically recited therein,but that there is no bar on additional features that are not recitedbeing present as well. The term “comprising” leaves the claim open forthe inclusion of unspecified ingredients even in major amounts. The term“consisting essentially of” in a claim means that the inventionnecessarily includes the listed ingredients, and is open to unlistedingredients that do not materially affect the basic and novel propertiesof the invention. A “consisting essentially of” claim occupies a middleground between closed claims that are written in a closed “consistingof” format and fully open claims that are drafted in a “comprising’format”. These terms can be used interchangeably herein if, and when,this may become necessary.

Regarding disclosed ranges, the endpoints of all ranges directed to thesame component or property are inclusive and independently combinable(e.g., ranges of “up to about 25%, or, more specifically, about 5% toabout 20%,” is inclusive of the endpoints and all intermediate values ofthe ranges of “about 5% to about 25%,” etc.). Numeric ranges recited inthe specification and claims are inclusive of the numbers defining therange and include each integer within the defined range, as well as allsubranges within the overall range.

“An affinity ligand” for affinity chromatography refers to a chemicalmoiety, coupled to a stationary phase, that serves as a biospecificsorptive group.

“Host cell” refers to a cell used to express an endogenous orheterologous nucleic acid sequence encoding a target peptide,polypeptide, or protein of interest.

“Parent cell from which it is derived” refers to a cell that is modifiedto then serve as a host cell of the present invention. As a non-limitingexample, an E. coli parent cell can be a conventional E. coli K-12 cell.Further E. coli parent cells are disclosed below.

A host cell of the present invention can be “derived” from a parent cellby reducing the genome of the host cell compared to the genome of theparent cell from which the host cell is derived by: (a) deleting genesof the parent cell, for example by knockout mutation, or (b) modifyingthe genome of the host cell compared to the genome of the parent cellfrom which the host cell is derived, or (c) reducing or completelyinhibiting expression of genes of the host cell compared to expressionof these genes in the parent cell from which the host cell is derived,wherein genes that are deleted, modified, and/or the expression of whichis reduced or completely inhibited in the host cell code for peptides,polypeptides, or proteins that impair the chromatographic separationefficiency of a target recombinant (or non-recombinant) peptide,polypeptide, or protein expressed in the host cell. This improves thechromatographic separation efficiency, including separation capacity, oftarget recombinant or non-recombinant molecules expressed in the hostcell compared to that when such target molecules are expressed in theparent cell, i.e., wherein genes that are deleted, modified, and/or theexpression of which is reduced or completely inhibited in the host cellare not deleted, not modified, or the expression of which is not reducedor completely inhibited in the host cell. As discussed below,identification of genes to be deleted, etc., from parent cells toproduce host cells can be accomplished by employing importance equation3. Host cells of the present invention exhibit growth rates and/orviability and/or capacity for expression of target molecules in therange of from about 60% to about 100%, or more; or from about 70% toabout 100%, or more; or from about 75% to about 100%, or more, of thatof the parent cells from which they are derived, and improvement inseparation efficiency, including separation capacity, of targetmolecules in the range of from about 5% to about 35%, or more; fromabout 5% to about 40%, or more; from about 5% to about 45%, or more; orfrom about 5% to about 50%, or more compared to that of the parent cellsfrom which they are derived. Host cells of the present invention cancomprise, consist essentially of, or consist of the gene deletions, genemodifications, and/or inhibited genes disclosed herein.

The phrase “a target recombinant therapeutic peptide, polypeptide, orprotein” and the like refers to a peptide, polypeptide, or proteinexhibiting human or veterinary medicinal properties, expressed usingrecombinant nucleic acid methodology. As used herein, “medicinalproperties” broadly includes not only medical therapeutic applications,but use for nutritional purposes and personal care as well.

The phrases “target host cell peptide, polypeptide or protein” or“endogenous target peptide, polypeptide or protein” and the like referto a peptide, polypeptide, or protein native to a host cell. In variousembodiments disclosed herein, such peptides, polypeptides, and proteinscan be expressed in host cells either naturally, e.g., under the controlof endogenous regulatory elements such as naturally occurring promoters,etc., without genetic manipulation, or by using recombinant nucleic acidmethodology to improve expression levels, e.g., by replacing naturalpromoters with stronger ones.

Thus, it should be noted that embodiments of the present invention,including all the parent cells, host cells, methods, etc., disclosedherein, are applicable not only to the expression and purification oftarget host cell or endogenous target peptides, polypeptides, orproteins via recombinant methods, but also to the expression andpurification of such peptides, polypeptides, and proteins that arenaturally expressed within host cells, i.e., without the application ofrecombinant methodology.

The phrase “heterologous target recombinant peptide, polypeptide orprotein” and the like refers to a peptide, polypeptide, or protein notnative to a host cell, which is expressed in such cell using recombinantnucleic acid methodology.

Heterologous nucleic acid fragments encoding such peptides, etc., suchas coding sequences that have been inserted into a host cell, are notnormally found in the genetic complement of the host cell. As usedherein, the term “heterologous” also refers to a nucleic acid fragmentderived from the same host cell, but which is located in a different,e.g., non-native, location within the genome of this cell. Thus, thecell can have more than the usual number of copy(ies) of such fragmentlocated in its(their) normal position within the genome. Heterologousnucleic acid fragments encoding recombinant peptides, polypeptides, orproteins of interest in plant cells can be expressed within differentgenomes within such cells, for example in the nuclear genome and withina plastid or mitochondrial genome. A nucleic acid fragment that isheterologous with respect to a cell into which it has been inserted ortransferred is sometimes referred to as a “transgene.”

“Essential genes” are defined by Gerdes et al. (2003) J. Bacteriol.185(19):5673-84 as genes that are needed for cell viability when grownin LB broth. For example the enzymes in the first half of coremetabolism are all considered essential; however, if the growth mediumis fortified with citrate, which can directly enter the Krebs cycle, thegenes coding for the enzymes preceeding the Krebs cycle can be safelydeleted without detrimental effects on cell viability. When geneessentiality is defined as the genes that are needed for cell viabilitywhen grown in minimal M9+ glucose as in Patrick et al. (December, 2007)Mol Biol Evol. 24(12):2716-22. Epub 2007 Sep. 19, all of the genes thatcode for enzymes in amino acid synthesis are considered essential;however, if the growth medium is fortified with these amino acids, thesegenes can also safely be deleted. These feeding strategies can be usedin the present host cells and methods to circumvent potentiallydeleterious effects due to deletion, modification, and/orreduction/inhibition of expression of essential genes that wouldotherwise adversely impact chromatographic separation efficiency ifpresent.

The phrase “a modified genome compared to the genome in the parent cellfrom which it is derived” refers to modification of genes to abate theundesirable effect(s) of the gene products on separation efficiencyperformed by, for example, point mutation, amino acid substitution,isozyme substitution, transposon mutagenesis, etc. As indicated,modification includes gene substitution. One example of genemodification to improve IMAC chromatography is to delete histidineresidues on the surface of interfering proteins when possible. In ionexchange chromatography, one could reduce the binding affinity ofnuisance proteins by altering amino acids to change protein surfacecharges. Modification also includes changes to essential genes thatinterfere with chromatographic separation efficiency by, for example,reducing their expression by replacing their naturally occurringpromoters with weaker promoters, introducing strategic point mutationsto replace amino acids involved in resin binding while still maintainingsatisfactory levels of gene/protein activity, or replacing endogenous E.coli genes with genes from other organisms that perform the same orsimilar functions and that do not significantly adversely affectchromatographic separation efficiency and separation capacity, and cellgrowth, viability, and capacity for expression, rather than deletingthem entirely. Such replacement genes include heterologs, homologs,analogs, paralogs, orthologs, and xenologs. These strategies facilitateimprovements in chromatographic separation efficiency even wheninterfering host cell proteins include essential genes.

“Proteome” refers to a collection of identifiable proteins expressed bya host cell.

“Chromatotome” refers to a proteome defined by a set of host cellproteins that bind a chromatographic stationary phase.

“Separatome” refers to a proteome defined by a set of host cell proteinsthat are associated with a separation technique (not limited to packedbed chromatography).

“Metalloproteome” refers to a proteome with the identifyingcharacteristic of interaction with metals or metal ions.

“Metabolome” refers to a collection of small-molecule metabolites likeglucose-6-phosphate and other molecules of similar molecular weight.

“Separation efficiency” is manifested as separation capacity, separationselectivity, or both. In many cases, separation capacity is a moreimportant parameter for the practice of the present invention.

“Separation capacity” refers to the amount of peptides, polypeptides,and/or proteins that can be captured during the loading cycle of achromatographic separation. Separation capacity is defined as the amountof target recombinant peptide, polypeptide, or protein adsorbed by acolumn per mass lysate fed to the column. The present inventionencompasses increases in separation capacity in the range of from about5% to about 35%, or more, for example from about 5% to about 40%, ormore; from about 5% to about 45%, or more; from about 5% to about 50%;or more, and so on similarly, depending on the number and particularcombination of genes deleted, etc. Such increases reflect an advantageof the present separatome invention concept over the separationcapacities achievable using standard host cells and extraction andpurification methods. i.e., compared to chromatographic separationcapacity of standard host cells that retain the presence of allnaturally occurring peptides, polypeptides, or proteins coded for bygenes that are deleted, modified, or the expression of which is reducedor completely inhibited in host cells of the present disclosure uponaffinity or adsorption, non-affinity column chromatography of targetrecombinant peptides, polypeptides, or proteins.

While “separation capacity” is discussed above with reference to atarget recombinant peptide, polypeptide, or protein, those of ordinaryskill in the art will recognize that this term also more generallyrefers to potential improvements in chromatographic separationefficiency, including separation capacity, by the deletion, etc., ofhost cell peptides, polypeptides, and proteins that would interfere withthe chromatographic purification of target peptides, etc., in theabsence of expression of any particular target. Therefore. “separationcapacity” can also be defined in terms of the amount of host cellpeptides, polypeptides, or proteins adsorbed by a column per mass lysatefed to the column. Viewed from this perspective, it is clear thatreducing interfering host cell peptides, polypeptides, or proteins thatbind the column results in an increase in separation capacity. In theabsence of expression of a particular target molecule, the potentialimprovement in chromatographic separation capacity is in the range offrom about 5% to about 35%, or more, for example from about 5% to about40%, or more; from about 5% to about 45%, or more, from about 5% toabout 50%, or more, and so on similarly, depending on the number andparticular combination of genes deleted, etc., upon affinity oradsorption, non-affinity column chromatography of the target molecule.Example 3 below demonstrates this principle by showing improvedseparation capacity in a modified E. coli host cell in the absence ofexpression of a target recombinant molecule. This example reflects theuniversal nature and applicability of the present host cells and methodsin peptide, polypeptide, or protein purification by affinity oradsorption, non-affinity column chromatography.

In both cases, separation efficiency, including “separation capacity”,is improved by the deletion, etc., of interfering host cell peptides,polypeptides, and proteins having a binding strength similar to that ofthe target recombinant or non-recombinant peptide, polypeptide, orprotein and/or that broadly elute as % B increases.

“Separation selectivity” refers to the amount of target protein/totalprotein captured by a chromatographic adsorbent. Separation selectivityis defined as the amount of target recombinant peptide, polypeptide, orprotein adsorbed by the column per total protein adsorbed to the column.

“Total Contaminant Pool (TCP)” refers to proteins that are known to bindto a given chromatography resin at a given pH. These proteins take upcolumn capacity. Elimination or reduction of such proteins results incolumn capacity improvement.

“Eluting Contaminant Pool (ECP)” refers to proteins that are part of theTCP, but which are further grouped by their elution conditions. Forexample, the proteins that would co-elute with the target protein init's specific elution window. Elimination or reduction of such proteinssimplifies purification and improves target protein purity.

“HCPs” refers to host cell proteins.

“Percent B” refers to a proportion or amount, expressed as a numberbetween 0 and 100%, of a mixture fed to a chromatography columncomprised of a blend of two fluids of different compositions, i.e.,composition A and composition B. A is the loading buffer, while B is theelution buffer. Percent B=100%−% A. As % B increases, the change inmobile phase composition causes proteins to be eluted in a differentialfashion, beginning with those of low affinity.

“Strongly retained” refers to peptides, polypeptides, and proteins thatelute from a chromatography column upon desorption due to stringentchanges in mobile phase composition identified by “percent B”.

“Weakly retained” refers to peptides, polypeptides, and proteins thatelute from a chromatography column upon desorption due to small changesin mobile phase composition identified by “percent B”.

“Common salt” refers to a compound that dissociates in water to form acation and an anion, such as a chloride salt, a sulfate salt, an acetatesalt, a carbonate salt, a propionate salt, etc., as would be apparent toone of ordinary skill in the art. Common cations in such salts are, forexample, sodium, potassium, and ammonium cations.

The phrase “chromatographically relevant host cell peptides,polypeptides, or proteins for column affinity chromatography or columnadsorption-based, non-affinity chromatography” refers to proteins of aseparatome or chromatotome.

“Importance” or “importance score” refers to the degree to which, shoulda host cell peptide, polypeptide, or protein be deleted, modified, orinhibited, capacity recovery is impacted. Proteins of chromatographicrelevance are considered important should large gains in capacityrecovery be achieved through deletion, modification, and/or inhibition.“Important” proteins are therefore a subset of relevant proteins.

“Reduced” in the context of the level of expression of peptides,polypeptides, or proteins from host cell genes (HCPs) refers todiminution in the amount of such expression products in the range offrom about 5% reduction to about 95% reduction, or more; from about 10%reduction to about 95% reduction, or more; or from about 25% reductionto about 95% reduction, or more, compared to the level of such productsnormally present in parent cells from which such host cells are derived.

“Scoring” or “importance scoring” refers to rank ordering members of aseparatome to identify host cell peptides, polypeptides, or proteinsthat impair the chromatographic separation efficiency of a targetrecombinant peptide, polypeptide, or protein expressed in the host cell,and to establish quantitative improvements gained through theirelimination.

“Operational variable” refers to a condition or operating parameter thatleads to different Damkohler, Biot, or Peclet numbers used to describe aseparation technique.

“Purify, purifying, purified” and the like refer to the process by whicha peptide, polypeptide, or protein in a mixture is enriched so as tocontain lesser amounts of materials derived from the host cell in whichit is expressed, and the enriched product, respectively.

“Plant cells” includes cells of flowering and non-flowering plants, aswell as algal cells, for example Chlamydomonas, Chlorella, etc.

Certain claims have unique formulae to mathematically define thenon-metabolic aspects of the separatome, with specific regard to theoverall impact a peptide, polypeptide, or protein has on columnefficiency. A peptide, polypeptide, or protein elutes or emerges from acolumn as a peak of material, first at low concentration increasing to amaximum value, then decreasing back to zero, in the characteristic shapeof a bell-like curve. The peak adopts a shape that may be described assharp/narrow, with the majority of material of interest contained in afew fractions; broad/shallow, with the majority of material present inmultiple fractions; or something in between. The time (retention time)at which the peak emerges is governed by binding strength. Peptides,polypeptides, and proteins with high affinity towards a ligand requiremore stringent conditions for desorption to occur, whereas those withlow affinity pass through the column unretained. The ability to captureboth phenomena, namely peak shape and retention time, is important toquantitatively establish the chromatographic relevance of a peptide,polypeptide, or protein. Once the relevance for a set of peptides,polypeptides, or proteins is established, molecular biology techniquesare then used to delete, modify, inhibit the expression of, orsubstitute genes associated with these interfering molecules to directlyincrease column capacity and indirectly enhance purity.

Defining “recovery potential” for protein (i) first involves determiningthe fractional capacity occupied by this particular host cell proteinby:

recovery potential_(i) =h _(i,total) /h _(total,ms)  Equation 1

with h_(total,ms)=total amount of host cell proteins bound to column,and h_(i,total)=the bound amount attributed to (i). The value ofrecovery potential is bound by zero and one, with a value of oneindicative of a single host cell protein, if removed from theseparatome, would achieve complete recovery of the column capacity.Extending this argument to the removal of (n) proteins, the term“capacity recovery” is defined in general as:

$\begin{matrix}{{{capacity}\mspace{14mu} {recovery}} = {100\% \mspace{11mu} x{\sum\limits_{i = 1}^{n}\; {{recovery}\mspace{14mu} {potential}_{i}}}}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

where the sigma operator allows one to sum the individual contributionsfor the set of (n) proteins. In the equation, n refers to number ofproteins, and i is an individual protein.

These two simple relationships provide the starting point to define howmuch capacity can be gained as genes are deleted, modified, inhibited,or substituted. The relationships do not, however, establish order orpriority within the context of peak shape and retention time. The latteris important to the disclosed invention because as mentioned previously,it is desired to focus efforts on common, problematic host cell proteinsrather than those that are specific to a target recombinant product.Strongly retained or high affinity host cell proteins that are bound andthat subsequently reduce column capacity would be generally problematicdue to their persistent presence. Other qualifiers generally regarded asproblematic would include high molecular weight (steric effects at highloading), sensitivity to proteolysis (multiple peaks or broad peak for asingle protein), and propensity for subunit adsorption (multiple peaksor broad peak for a single protein). A criterion has been developed toscore and rank the “importance” of a protein (i) within a separatome,i.e., the “importance score” (IS), namely:

$\begin{matrix}{{importance}_{i} = {\Sigma_{j}\left\lbrack {{b_{1}\left( \frac{y_{c_{j}}}{y_{\max}} \right)}\left( \frac{h_{i,_{j}}}{h_{i,{total}}} \right)\left( \frac{h_{i,_{j}}}{h_{j,{total}}} \right)\left( \frac{{MW}_{i}}{{MW}_{ref}} \right)^{\alpha}} \right\rbrack}_{i}} & {{Equation}\mspace{14mu} 3}\end{matrix}$

with the following definitions: b₁=scaling parameter, y_(cj) andy_(max)=concentration of mobile phase eluent in fraction (j) and maximumvalue, respectively; h_(i,j) and h_(i,total)=the amount of protein (i)in fraction (j) and total bound protein (i), respectively;h_(j,total)=total amount of protein in fraction (j); MW_(i)=molecularweight of protein (i); MW_(ref)=molecular weight of a reference proteinwithin the separatome; α=steric factor, and i=protein. These ratioterms—the y's and h's—adopt values between 0 and 1, yet hold differentsignificances. A protein that remains bound and requires stringentconditions for elution reflects a y ratio to be close to, if not equalto, unity. A protein that emerges as a tight peak presents with a ratiofor h close to unity, and finally, should that emerging peak constitutethe majority of fraction (j), the third ratio would be close to unity.Multiplying each ratio, and summing the product of these ratios for eachfraction (j) where (i) is present provides a quantitative ranking. Forexample, a protein that is retained at high NaCl concentration andemerges as a sharp peak would be deemed chromatographically relevant andwill be scored as high with this formula. A second example would be aprotein that broadly elutes. It would also receive a high score orrelevancy because its score would be high by virtue of its presence inmultiple fractions.

Lastly, there requires a consideration of steric effects. As achromatography column becomes loaded, larger proteins interact withmultiple ligands either directly through adsorption, or indirectlythrough hindrance of binding. When steric effects require consideration,the basic equation contains a molecular weight ratio raised to a powerthat is descriptive of these phenomena. A unitless, non-zero alpha inthe above equation, with a preferred value between 0 and 1, wouldindicate some degree of steric effects. Note that the general form ofthe importance equation also permits scale-parameters (b₁) to adjust theweighting of a particular score. For example, b₁ may be used to indicatemetabolic necessity (b₁=0), meaning a zero value will force a low scorebecause it likely will not be deleted from the genome, b₁ is unitless,and adopts a value between 0 and 1. Upon ranking the proteins in aseparatome, the essentiality of a protein is determined by reference tothe E. coli literature, e.g., Gerdes et al. (2003) J. Bacteriol.185(19):5673-84. Strategies for circumventing deletion of essentialproteins are described herein.

The importance score is defined in this fashion because this empiricallyderived equation captures the characteristics of both binding andelution data without solving numerical models of multi-component liquidchromatography to define the association and dissociation rateconstants. Molecular weight is included in the importance score since itplays a role when the column is under fully loaded or breakthroughloading conditions. This ratio is raised to α, where the α term accountsfor column saturation, wherein when the column is fully saturated α=1and when the column is unsaturated α<1. This causes the MW term to bedropped in cases where the column is not fully saturated, and thusmolecular weight, or the approximate size, of the protein do not factorinto overall column capacity. In the equation, the ratios of y's and h'sadopt values between 0 and 1. A protein that remains bound and requiresstringent conditions for elution exhibits a ratio

$\left( \frac{y_{c_{j}}}{y_{\max}} \right)$

close to, or equal to, unity, whereas a protein that emerges as a tightpeak has

$\left( \frac{h_{i,_{j}}}{h_{i,{total}}} \right)$

ratio close to unity. Finally, a

$\left( \frac{h_{i,j}}{h_{j,{total}}} \right)$

ratio is close to unity if it constitutes the majority of fraction (j).

Importance score values are between 0 and 1. “Rank” of proteinsdetermined according to Equation 3 are relative values compared to oneanother.

To summarize, the basic form of the equation favors the elimination ordeletion of peptides, polypeptides, or proteins that have high affinitytoward the adsorbent and/or broadly elute as % B increases, with somedegree of freedom to permit the tailoring of the modifications shouldthe host cell be expressly used for a single recombinant DNA product andnot a variety of products.

Commercially Important Protein Products

Exemplary, non-limiting, commercially important peptide, polypeptide,and protein products that can be expressed, recovered, and purifiedusing the host cells, methods, and separatome information disclosedherein include, but are not limited to, the following.

Therapeutic Proteins

Examples of therapeutic human proteins that have been synthesized fromgenes cloned in bacteria and/or eukaryotic cells, or by expression inplants or animals, include antibodies and antigen-binding fragments;vaccines; al-Antitrypsin (emphysema); deoxyribonuclease (cysticfibrosis); epidermal growth factor (ulcers); erythropoietin (anemia);Factor VIII (hemophilia); Factor IX (Christmas disease); fibroblastgrowth factor (ulcers); follicle stimulating hormone (infertilitytreatment); granulocyte colony stimulating factor (cancers); insulin(diabetes); insulin-like growth factor 1 (growth disorders);interferon-α (leukemia and other cancers); interferon-β (cancers, AIDS);interferon-γ (cancers, rheumatoid arthritis); interleukins (cancers,immune disorders); lung surfactant protein (respiratory distress);relaxin (aid in childbirth); serum albumin (plasma supplement);somatostatin (growth disorders); somatotrophin (growth disorders);superoxide dismutase (free radical damage in kidney transplants); tissueplasminogen activator (heart attack); tumor necrosis factor (cancers).

Proteins and Enzymes Used in Analytical Applications

In addition to the use of antibodies and enzymes as therapeutic agents,they are also used in the diagnosis of diseases as the components ofsome confirmatory tests of certain diagnostic procedures. Hexokinase andglucose oxidase are used in the quantification of glucose in the serumand urine. Glucose-oxidase is used in glucose electrodes. Uricase isused for the estimation of uric acid present in urine. Alkalinephosphatase, horseradish peroxidase, and antibodies are used in ELISA(Enzyme Linked Immunosorbent Assay).

Industrial Enzymes and Proteins

Industrially useful enzymes include carbohydrate-hydrolyzing enzymessuch as amylases, cellulase, invertases, etc.; proteolytic enzymes suchas papain, trypsin, chymotrypsin, etc.; and other bacterial andfungal-derived proteolytic enzymes and lipases that can hydrolyzevarious types of lipids and fats. All these enzymes are important in thefood and beverage industries, the textile industry, paper industry, anddetergent industry. Proteases have a special use in the beverageindustry, meat and leather industries, cheese production, detergentindustry, bread, and confectionery industry. Various types of lipasesare used for the modifications of various types of lipids and fats,production of various organic acids including fatty acids, indetergents, and production of cocoa butter. In addition to all these,enzymes are used in chemical industries as reagents in organic synthesisfor carrying out stereospecific reactions.

Depending on the intended use, proteins and enzymes can be employed invarying degrees of purity, i.e., highly purified preparationsapproaching nearly 100% purity are not always required, and thereforeextensive “polishing” chromatographic steps may not be required afterinitial purification. Such additional steps can therefore be consideredoptional for particular applications.

Non-Catalytic Functional Proteins

These commercially important proteins are used in the food industry asemulsifiers, for inducing gelation, water binding, foaming, whipping,etc. These non-catalytic functional proteins are classified as wheyproteins. The proteins that remain in solution after the removal ofcasein are by definition called whey proteins.

Commercially available whey protein concentrates contain 35% to 95%protein. If they are added to food on a solid's basis, there will belarge differences in functionality due to the differences in proteincontent. Most food formulations call for a certain protein content andthus whey-protein concentrates are generally utilized as a constantprotein base. In this case, the differences due to protein content assuch should be eliminated. As the protein content increases, thecomposition of other components in the whey-protein concentrate mustalso change and these changes in composition have an effect onfunctionality.

Nutraceutical Proteins

Nutraceutical proteins represent a class of nutritionally-importantproteins having therapeutic activity. The whey-protein concentrates andsome of the milk proteins of infant foods contain certain pharmaceuticalproteins having high nutritive quality. Infants get the requiredproteins from the mother's milk, which also contains certain therapeuticproteins that protect the baby from infection and other problems. Thereare other infant foods, which also have more or less the samecomposition as that of mother's milk, made up of cow's and buffalo'smilk. All these food proteins provide the infants the raw buildingmaterials in the form of essential amino acids and at the same timeprotect them from microbial infections and other diseases.

Large Scale Enzyme Applications

Detergents

Bacterial proteinases are still the most important detergent enzymes.Lipases decompose fats into more water-soluble compounds. Amylases areused in detergents to remove starch based stains.

Starch Hydrolysis and Fructose Production

The use of starch degrading enzymes was the first large scaleapplication of microbial enzymes in food industry. Mainly two enzymescarry out conversion of starch to glucose: alpha-amylase and fungalenzymes. Fructose is produced from sucrose as a starting material.Sucrose is split by invertase into glucose and fructose, and fructose isseparated and crystallized.

Beverages

Enzymes have many applications in the beverage industry. Lactase splitsmilk-sugar lactose into glucose and galactose. This process is used formilk products that are consumed by lactose intolerant consumers.Addition of pectinase, xylanase, and cellulase improve the liberation ofthe juice from pulp. Similarly, enzymes are widely used in wineproduction.

Textiles

The use of enzymes in the textile industry is one of the most rapidlygrowing fields in industrial enzymology. The enzymes used in the textilefield are amylases, catalase, and lactases, which are used to removestarch, degrade excess hydrogen peroxide, bleach textiles, and degradelignin.

Animal Feed

Addition of xylanase to wheat-based broiler feed has increased theavailable metabolizable energy 7-10% in various studies. Enzyme additionreduces viscosity, which increases absorption of nutrients, liberatesnutrients either by hydrolysis of non-degradable fibers or by liberatingnutrients blocked by these fibers, and reduces the amount of feces.

Baking

Alpha-amylases have been most widely studied in connection with improvedbread quality and increased shelf life. Use of xylanases decreases thewater absorption, and thus reduces the amount of added water needed, inbaking. This leads to more stable dough. Proteinases can be added toimprove dough-handling properties; glucose oxidase has been used toreplace chemical oxidants and lipases to strengthen gluten, which leadsto more stable dough and better bread quality.

Pulp and Paper

The major application in the pulp and paper industry is the use ofxylanases in pulp bleaching. This considerably reduces the need forchlorine based bleaching chemicals. In paper making, amylase enzymes areused especially in modification of starch. Pitch is a sticky substancepresent mainly in softwoods. Pitch causes problems in paper machines andcan be removed by lipases.

Leather

The leather industry uses proteolytic and lipolytic enzymes in leatherprocessing. Enzymes are used to remove unwanted parts. In dehairing anddewooling phases, bacterial proteases are used to assist the alkalinechemical process. This results in a more environmentally friendlyprocess and improves the quality of the leather. Bacterial and fungalenzymes are used to make leather soft and easier to dye.

Specialty Enzymes

There are a large number of specialty applications for enzymes. Theseinclude the use of enzymes in analytical applications, flavorproduction, protein modification, personal care products,DNA-technology, and in fine chemical production.

Enzymes in Analytics

Enzymes are widely used in clinical analytical methodology. Contrary tobulk industrial enzymes, these enzymes need to be free from sideactivities. This means that elaborate purification processes are needed.

An important development in analytical chemistry is biosensors. The mostwidely used application is a glucose biosensor involving glucose oxidasecatalyzed reaction. Several commercial instruments are available whichapply this principle for measurement of molecules like glucose, lactate,lactose, sucrose, ethanol, methanol, cholesterol, and some amino acids.

Enzymes in Personal Care Products

Personal care products are a relatively new area for enzymes. Proteinaseand lipase containing enzyme solutions are used for contact lenscleaning. Hydrogen peroxide is used in disinfections of contact lenses.The residual hydrogen peroxide after disinfections can be removed bycatalase. Some toothpaste contains glucoamylase and glucose oxidase.Enzymes are also being studied for applications in skin and hair careproducts.

Enzymes Used in DNA-Technology

DNA-technology is an important tool in the enzyme industry. Mosttraditional enzymes are produced by organisms that have been geneticallymodified to overproduce desired enzymes. Recombinant DNA methodology hasbeen used to engineer overproducing microorganisms, and employs enzymessuch as nucleases (especially restriction endonucleases), ligases,polymerases, and DNA-modifying enzymes to modify genes and constructnecessary expression cassettes and vectors.

Enzymes in Fine Chemical Production

In spite of some successes, commercial production of chemicals by livingcells via pathway engineering is still in many cases the bestalternative to apply biocatalysis. Isolated enzymes have, however, beensuccessfully used in fine chemical synthesis. Some of the most importantexamples are:

Chirally Pure Amino Acids and Aspartame

Natural amino acids are usually produced by microbial fermentation.Novel enzymatic resolution methods have been developed for theproduction of L- and D-amino acids. Aspartame, the intensive non-caloriesweetener, is synthesized in non-aqueous conditions by thermolysin, aproteolytic enzyme.

Rare Sugars

Recently, enzymatic methods have been developed to manufacturepractically all D- and L-forms of simple sugars. Glucose isomerase isone of the important industrial enzymes used in fructose manufacturing.

Semisynthetic Penicillins

Penicillin is produced by genetically modified strains of Penicilliumstrains. Most of the penicillin is convened by immobilized acylases to6-aminopenicillanic acid, which serves as a backbone for manysemisynthetic penicillins.

Lipase-Based Reactions

In addition to detergent applications, lipases can be used in versatilechemical reactions since they are active in organic solvents. Lipasesare used in transesterification, for enantiomeric separation ofalcohols, and for the separation of racemic mixtures. Lipases have alsobeen used to form aromatic and aliphatic polymers.

Enzymatic Oligosaccharide Synthesis

The chemical synthesis of oligosaccharides is a complicated multi-stepeffort. Biocatalytic syntheses with isolated enzymes likeglycosyltransferases and glycosidases or engineered whole cells arepowerful alternatives to chemical methods. Oligosaccharides have foundapplications in cosmetics, medicines and as functional foods.

OVERVIEW

Disclosed herein is a separatome-based host cell peptide, polypeptide,and protein expression and purification platform focusing on theproteomes of various chromatographic methods to provide a single hostcell line, or set of host cell lines, that can be used for expression ofa wide variety of recombinant peptides, polypeptides, and proteins,thereby eliminating the need to develop individual host cell lines foreach purification process.

The “separatome” of the present separatome-based protein expression andpurification platform involves the juxtaposition of the bindingproperties of host cell peptides, polypeptides, and proteins in commonchromatographic techniques (e.g., IMAC. IEX, and/or HIC) with thecharacteristics and location of the corresponding encoding genes on thetarget host cell chromosome(s). While the examples of theseparatome-based protein expression and purification platform disclosedherein focus on Escherichia coli as the host cell, and its chromatotome,the invention is not limited thereto as the separatome-based peptide,polypeptide, and protein expression and purification platform can extendto any suitable host conventionally used for recombinant expression,such as Lactococcus lactis. Bacillus species such as B. licheniformis,B. amyloliquefaciens, and B. subtilis, Corynebacterium glutamicum,Pseudomonas fluorescens, or other prokaryotes; fungi, including variousyeasts such as Saccharomyces cerevisiae, Pichia (now K.) pastoris, andPichia methanolica; insect cells; mammalian cells; plant cells,including for example, tobacco (e.g., cultivars BY-2 and NT-1), alfalfa,rice, tomato, soybean, as well as algal cells; and protozoal cells suchas the non-pathogenic strain of Leishmania tarentolae, etc.

The present separatome-based peptide, polypeptide, and proteinexpression and purification platform is an efficient bioseparationsystem that intertwines host cell strain and chromatography. Since thehigh cost of product purification often limits the availability oftherapeutic proteins of interest to immunology, vaccine development,pharmaceutical production, and diagnostic reagents, as well as theavailability of enzymes for various applications, the presentseparatome-based peptide, polypeptide, and protein expression andpurification platform provides alternative pathways towards efficientpurification based on the utilization of proteome data. In particular,the separatome-based protein expression and purification platformprovides for: (i) a system of chromatographic data based on identified,conserved genomic regions that span resin- and gradient-specificchromatographies, or chromatotomes, for example, a database of E. coliproteins that span the chromatography total contaminant pool(TCP)/elution contaminant pool (ECP) and bind under various conditionsto a variety of chromatographic resins; (ii) a process to minimizecontaminant pools of nuisance or coeluting proteins associated withspecific chromatographies, for example, gradients that substantiallydecrease the number of coeluting proteins encountered duringbioseparation, and the specific, targeted deletion of nuisance host cellpeptide-, polypeptide-, and protein-encoding genes to minimizecontaminant pools associated with affinity adsorption and non-affinityadsorption chromatographies, including IMAC, cation IEX, anion IEX, HIC,and combinations thereof.

The separatome-based peptide, polypeptide, and protein expression andpurification platform is constructed based upon a computer system ofidentified, conserved genomic regions that span resin- and gradientspecific-chromatographies, or chromatotomes. The computer systemincludes a data visualization program/application resident on a standardcomputer device, such as a mainframe, desktop, or other computer. Forexample, the computer may have a central processor that controls theoverall operation of the computer and a system bus that connects thecentral processor to one or more conventional components, such as anetwork card or modem. The computer may also include a variety ofinterface ports and drives for reading and writing data or files. A userof the separatome-based protein expression and purification platform caninteract with the computer with a keyboard, pointing device, microphone,pen device, or other input device. The computer may be connected via asuitable network connection, such as a T1 line, a common local areanetwork (“LAN”), via the worldwide web, or via other mechanism forconnecting computer devices.

The separatome-based peptide, polypeptide, and protein expression andpurification platform will utilize large amounts of data compiled on themetalloproteome and metabolome of the selected host cell, such as E.coli. The data visualization program/application, such as Circos®, asoftware package for visualizing data and information in a circularlayout (available from Canada's Michael Smith Genome Sciences Center),enables the user to visualize the large amounts of data and informationfor exploring relationships between objects or positions. FIGS. 1 and 2illustrates examples of how the data visualization program/applicationcould illustrate the E. coli chromosome mapped with the chromatotome ofmultiple chromatographic techniques, thus showing where the differentchromatotomes lie within the greater genome. Each line in FIG. 1represents a single contaminating protein, and the graph at its baseshows the total concentration of the protein as a percent of the TCP orECP. If each TCP is subdivided into its respective ECPs, then furthercorollaries can be drawn between proteins and genomic location. Further,segments of the ring represent the E. coli genome or the proteomeassociated with a particular isolation technique. With respect to E.coli, inner rings can represent additional information likeessentiality, successful deletion, metabolic function, etc. For a givenchromatographic technique, inner ring data can represent conditions thattrigger adsorption or elution, concentration in the extract, and if thisprotein is differentially expressed during stress.

In addition, the separatome-based peptide, polypeptide, and proteinexpression and purification platform may utilize and/or incorporate dataabout the target genome and proteome sequences, such as from Ecogene®(Institute for Advanced Biosciences, Keio University and IntegratedGenomics, Chicago, Ill.). The data visualization program/application ofthe separatome-based protein expression and purification platformprovides the user a feasible means of utilizing the data by melding itinto a productive format, and in particular, the data visualizationprogram/application provides the ability to visually summarize largecollections of data covering peptides, polypeptides, and proteinsencountered in the chromatotome and their essentiality.

The mapping and plotting of the IMAC, IEX and HIC data by theseparatome-based peptide, polypeptide, and protein expression andpurification platform allows for the identification of large contiguousregions of contaminants from several chromatography techniques that maybe targeted for modification if necessary.

Since the overall structure of a target recombinant peptide,polypeptide, or protein and the column resin are usually fixedconstraints, a reduction in contaminant species has the ability toimprove chromatographic recovery and purification via elimination ofundesirable binding events. Overall reduction of contaminant species,including undesired host cell peptides, polypeptides, and proteins, canbe achieved by removal, modification, or inhibition of the expression ofthe genomic regions coding for the contaminants.

General Methods

Practice of the various embodiments of the present invention employs,unless otherwise indicated, conventional techniques of molecularbiology, recombinant DNA technology, microbiology, chemistry, etc.,which are well known in the art and within the capabilities of those ofordinary skill in the art. Such techniques include the followingnon-limiting examples: preparation of cellular, plasmid, andbacteriophage DNA; manipulation of purified DNA using nucleases,ligases, polymerases, and DNA-modifying enzymes; introduction of DNAinto living cells; cloning vectors for various organisms; PCR; genedeletion, modification, replacement, or inhibition; production ofrecombinant peptides, polypeptides, and proteins in host cells;chromatographic methods; etc.

Such methods are well known in the art and are described, for example,in Green and Sambrook (2012) Molecular Cloning: A Laboratory Manual,Fourth Edition, Cold Spring Harbor Laboratory Press; Ausubel et al.(2003 and periodic supplements) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y.; Amberg et al. (2005) Methods in YeastGenetics: A Cold Spring Harbor Laboratory Course Manual, 2005 Edition,Cold Spring Harbor Laboratory Press; Roe et al. (1996) DNA Isolation andSequencing: Essential Techniques, John Wiley & Sons; J. M. Polak andJames O′D. McGee (1990) In Situ Hybridization: Principles and Practice;Oxford University Press; M. J. Gait (Editor) (1984) OligonucleotideSynthesis: A Practical Approach, IRL Press; D. M. J. Lilley and J. E.Dahlberg (1992) Methods in Enzymology: DNA Structure Part A: Synthesisand Physical Analysis of DNA, Academic Press; and Lab Ref: A Handbook ofRecipes, Reagents, and Other Reference Tools for Use at the Bench,Edited by Jane Roskams and Linda Rodgers (2002) Cold Spring HarborLaboratory Press; Burgess and Deutscher (2009) Guide to ProteinPurification, Second Edition (Methods in Enzymology, Vol. 463), AcademicPress. Note also U.S. Pat. Nos. 8,178,339; 8,119,365; 8,043,842;8,039,243; 7,303,906; 6,989,265; US20120219994A1; and EP 1483367B1. Theentire contents of each of these texts and patent documents is hereinincorporated by reference.

Designations of E. coli genes change from time to time or are referredto by different names in different laboratories. For example, hldD isalso known as rfaD. Any discrepancies between the E. coli genedesignations disclosed herein and updated designations can beascertained from EcoCyc ([EcoCyc13] Keseler et al. (2013) EcoCyc: fusingmodel organism databases with systems biology. Nucleic Acids Research41:D605-612 and EcoGene (Zhou et al. (2013) EcoGene 3.0 Nucleic AcidsResearch, 41 (D1): D613-D624), which are curated E. coli databases wellknown in the art.

Methods for Deleting, Modifying, and Inhibiting the Expression of Genesin E. coli

-   Baba et al. (2006) Mol. Syst. Biol. 2:2006.0008,    doi:10.1038/msb4100050, discloses methods for making precisely    defined single gene deletions in E. coli.-   Datsenko et al. (2000) Proc. Natl. Acad. Sci. USA. 97(12):6640-5    discloses methods for inactivating chromosomal genes in E. coli    using PCR products.-   Stringer et al. (2012) PLoS ONE 7(9): e44841.    doi:10.1371/journal.pone.0044841 discloses a rapid, efficient,    PCR-based recombineering method that can be used to introduce    scar-free point mutations, deletions, epitope tags, and promoters    into the genomes of multiple species of enteric bacteria.-   Le Cong et al. (2013) Science 339:819-823; Jiang et al. (2013)    Nature Biotechnology 31(3):233-239; Mali et al. (2013) Nature    Methods 10(10):957-963: Sander et al. (2014) Nature Biotechnology    32(4):347-355; and U.S. Pat. No. 8,697,359 disclose CRISPR-Cas    systems for editing, regulating, and targeting genomes.-   Methods for RNA silencing and antisense oligonucleotide inhibition    of gene expression are well known in the art. Note, for example, the    reviews in Nature (2009) 457, No. 7228, pp. 395-433 and Molecular    Cancer Therapeutics (2002) 1:347-355, respectively.-   E. coli gene essentiality data can be retrieved from Gerdes et    al. (2003) J. Bacteriol. 185(19):5673-84) which compiles gene    essentiality from their own research as well as the Profiling of E.    coli Chromosome (PEC) database (Hashimoto et al. (2005) Molecular    Microbiology 55(1):137-49; Kato and Hashimoto (2007) Molecular    Systems Biology 3(132):132; and Kang Y et al. (2004) J. Bacteriol.    186(15):4921-30). Such data can also be determined empirically.

Frequently Used Expression Systems for Foreign Genes

-   Yin et al. (2007) Journal of Biotechnology 127(3):335-347 reviews    the most frequently used expression systems for foreign genes.-   Baneyx (1999) Curr. Opin. Biotechnol. 10(5): 411-21 describes    protein production in frequently used host cell systems.

Examples of specific E. coli parent and host cells useful in the presentinvention include the following. These listings should not be construedto be limiting as other E. coli host cells known in the art are alsouseful in embodiments of the present methods, and are encompassedherein.

TABLE 1 References Disclosing E. coli Strain Genomic Sequences TableGenome Entry E. coli Strain Reference Size Number Number (Source ofGenomic Sequence) (Mb) 1 E. coli K-12 Blattner FR, et al. Science 19974.639 Sep. 5; 277(5331): 1453-62. 2 E. coli MG1655 Blattner FR, et al.Science 1997 4.639 Sep. 5; 277(5331): 1453-62. 3 E. coli BL21 (DE3)Jeong Hm et al. J Mol Biol 2009 4.56 Dec. 11; 394(4): 644-52 4 E. coliDH10B Durfee et al. J Bacteriol. 2008 4.69 April; 190(7): 2597-606

As indicated in Table 1, E. coli strains useful in various embodimentsof the present invention include K-12 and B strains. C and W strains arealso useful. Derivatives and substrains of all of these strains, as areknown to those of ordinary skill in the art, are also useful.

Useful K-12 derivatives include, but are not limited to, strains such asW3110, DH10B, DH5alpha, DH1, MG1655, BW2952, and their derivatives.

Useful B strain derivatives include, but are not limited to, B REL606,BL21, BL21-DE3, and their derivatives.

Other useful E. coli strains include, but are not limited to, thefollowing, including their derivatives and substrains:

-   -   Alpha-Select Bacteriophage T1-Resistant Gold Efficiency (F− deoR        endA1 recA1 relA1 gyrA96 hsdR17(rk⁻, mk₊) supE44 thi-1 phoA        Δ(lacZYA-argF)U169 Φ80lacZΔM15λ−),    -   Alpha-Select Bacteriophage T1-Resistant Silver Efficiency (F−        deoR endA1 recA1 relA1 gyrA96 hsdR17(rk⁻, mk₊) supE44 thi-1 phoA        Δ(lacZYA-argF)U169 Φ80lacZΔM15λ−),    -   Alpha-Select Bronze Efficiency (F− deoR endA1 recA1 relA1 gyrA96        hsdR17(rk−, mk+) supE44 thi-1 phoA Δ(lacZYA-argF)U169        Φ80lacZΔM15λ−),    -   Alpha-Select (F− deoR endA1 recA1 relA1 gyrA96 hsdR17(rk−, mk+)        supE44 thi-1 phoA Δ(lacZYA-argF)U169 Φ80lacZΔM15λ−),    -   AG1 (endA1 recA1 gyrA96 thi-1 relA1 glnV44 hsdR17(r_(K) ⁻ m_(K)        ⁺)),    -   AB1157 (thr-1, araC14, leuB6(Am), Δ(gpt-proA)62, lacY1, tsx-33,        qsr′-0, glnV44(AS), galK2(Oc), LAM−, Rac-0, hisG4(Oc), rfbC1,        mgl-51, rpoS396(Am), rpsL31(strR), kdgK51, xylA5, mtl-1,        argE3(Oc), thi-1),    -   B2155 (thrB1004 pro thi strA hsdsS lacZD M15 (F′lacZD M15        lacI^(q) traD36 proA⁺proB⁺) Δ dapA::erm (Erm^(r)) pir::RP4        [::kan (Km^(r)) from SM10]),    -   B834(DE3) (F⁻ompT hsdS_(B)(r_(B) ⁻ m_(B) ⁻) gal dcm met (DE3)),    -   BIOBlue (recA1 endA1 gyrA96 thi-1 hsdR17(rk−, mk+) supE44 relA1        lac [F′ proAB lacI^(q)ZΔM15 Tn10(Tet^(r))]),    -   BL21 (E. coli B F− dcm ompT hsdS(r_(B)− m_(B)−) gal        [malB⁺]_(K-12)(λ^(S))),    -   BL21(AI) (F⁻ ompT gal dcm lon hsdS_(B)(r_(B) ⁻ m_(B) ⁻)        araB::T7RNAP-tetA),    -   BL21(DE3) (F⁻ ompT gal dcm lon hsdS_(B)(r_(B) ⁻ m_(B) ⁻) λ(DE3        [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])),    -   BL21 (DE3) pLysS (F− ompT hsdSB(rB−, mB−) gal dcm (DE3) pLysS        (CamR)),    -   BL21-T1R (F− ompT hsdSB(rB− mB−) gal dcm tonA),    -   BNN93 (F⁻ tonA21 thi-1 thr-1 leuB6 lacY1 glnV44 rfbC1 fhuA1 mcrB        e14-(mcrA⁻) hsdR(r_(K) ⁻m_(K) ⁺) λ⁻)    -   BNN97 (BNN93 (λgt11)),    -   BW26434 (Δ(araD-araB)567, Δ(lacA-lacZ)514(::kan),        lacI^(p)-4000(lacI^(q)), λ⁻, rpoS396(Am)?, rph-1,        Δ(rhaD-rhaB)568, bsdR514),    -   C600 (F⁻ tonA21 thi-1 thr-1 leuB6 lacY1 glnV44 rfbC1 fhuA1λ⁻),    -   CAG597 (F⁻ lacZ(am) pho(am) lyrT[supC(ts)] trp(am) rpsL(Str^(R))        rpoH(am)165 zhg::Tn10 mal(am)),    -   CAG626 (F⁻ lacZ(am) pho(am) lon trp(am) tyrT[supC(ts)]        rpsL(Str^(R)) mal(am)),    -   CAG629 (F⁻ lacZ(am) pho(am) lon supC(ts) trp(am) rpsL        rpoH(am)165 zhg::Tn10 mal(am)),    -   CH3-Blue (F− ΔmcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1        endA1 ara Δ139 Δ(ara, leu)7697 galU galrpsL(Str^(R)) nupG λ−).    -   CSH50 (F⁻ λ⁻ ara Δ(lac-pro) rpsL thi fimE::IS1),    -   D1210 (HB101 lacI^(q) lacY⁺),    -   dam-dcm-Bacteriophage T1-Resistant (F− dam-13:Tn9(Cam^(R))dcm-6        ara-14 hisG4 leuB6 thi-1 lacY1 galK2 galT22 glnV44 hsdR2 xylA5        mtl-1 rpsL136(Str^(R)) rtbD1 tonA31 tsx78 mcrA mcrB1),    -   DB3.1 (F− gyrA462 endA1 glnV44 Δ(sr1-recA) mcrB mrr hsdS20(r_(B)        ⁻, m_(B) ⁺) ara14 galK2 lacY1 proA2 rpsL20(Sm^(r)) xy15 Δleu        mtl1),    -   DH1 (endA1 recA1 gyrA96 thi-1 glnV44 relA1 hsdR17(r_(K) ⁻ m_(K)        ⁺) λ⁻),    -   DH5α Turbo (F′ proA+B+ lacI^(q) Δ lacZ M15/fhuA2 Δ(lac-proAB)        glnV gal R(zgb-210::Tn10)Tet^(S) endA1 thi-1 Δ(hsdS-mcrB)5),    -   DH12S (mcrA Δ(mrr-hsdRMS-mcrBC) φ80d lacZΔM15 ΔlacX74 recA1 deoR        Δ(ara, leu)7697 araD139 galU galK rpsL F′ [proAB⁺        lacI^(q)ZΔM15]),    -   DM1 (F− dam-13::Tn9(Cm^(R)) dcm− mcrB hsdR-M+ gal1 gal2 ara−        lac− thr− leu− tonR tsxR Su0),    -   E. CLONI® 5ALPHA (fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80        Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17),    -   E. CLONI® 10G (F− mcrA Δ(mnr-hsdRMS-mcrBC) endA1 recA1        Φ80dlacZΔM15 ΔlacX74 araI139 Δ(ara,leu)7697galU galK rpsL nupG        λ− tonA (StrR)),    -   E. CLONI® 10GF′ ([F′ pro A+B+ lacI^(q)ZΔM15::T10 (Tet^(R))]/mcrA        Δ(mrr-hsdRMS-mcrBC) endA1 recA1 Φ80dlacZΔM15 ΔlacX74 araD139        Δ(ara, leu)7697 galU galK rpsL nupG λ− tonA (StrR)),    -   E. coli K12 ER2738 (F′proA+B+ lacI^(q) Δ(lacZ)M15        zzf::Tn10(Tet^(R))/fhuA2 glnV Δ(lac-proAB) thi-1 Δ(hsdS-mcrB)5),    -   ElectroMax™ DH10B (F⁻mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15        ΔlacX74 recA1 endA1 araD139Δ(ara,leu)7697 galU galK λ⁻rpsL        nupG),    -   ELECTROMAX™ DH5ALPHA-E (F− φ80lacZΔM15 Δ(lacZY A-argF) U169        recA1 endA1 hsdR17 (rk−, mk+) galphoA supE44λ-thi-1 gyrA96        relA1),    -   ElectroSHOX (F− mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74        recA1 endA1 ara Δ139 Δ(ara, leu)7697 galU galKrpsL(Str^(R)) nupG        λ⁻)    -   EP-MAX™10B F′ (mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74        deoR recA1 endA1 araD139 Δ(ara, leu)7697 galU galK rpsL nupG        λ−/F′[lacI^(q)ZΔM15 Tn10 (Tet^(R))]),    -   ER1793 (F⁻ fhuA2 Δ(lacZ)r1 glnV44 e14⁻(McrA⁻) trp-31 his-1        rpsL104 xyl-7 mtl-2 metB1 Δ(mcrC-mrr)114::IS10),    -   ER1821 (F⁻ glnV44 e14⁻(McrA⁻) rfbD1? rel4? endA1 spoT1? thi-1        Δ(mcrC-mrr))114::IS10),    -   ER2738 (F′proA⁺B⁺ lacI^(q) Δ(lacZ)M15 zzf::Tn10(Tet^(R))/fhuA2        glnV Δ(lac-proAB) thi-1 Δ(hsdS-mcrB)5),    -   ER2267 (F′ proA⁺B⁺ lacI^(q) Δ(lacZ)M15 zzf::mini-Tn10        (Kan^(R))/Δ(argF-lacZ)U169 glnV44 e14⁻(McrA⁻) rfbD1? recA1        relA1? endA1 spoT1? thi-1 Δ(mcrC-mrr)114::IS10),    -   ER2507 (F⁻ ara-14 leuB6 fhuA2 Δ(argF-lac)U169 lacY1 glnV44 galK2        rpsL20 xyl-5 mtl-5 Δ(malB)        zjc::Tn5(Kan^(R))Δ(mcrC-mrr)_(HB101)),    -   ER2508 (F⁻ ara-14 leuB6 fhuA2 Δ(argF-lac)U169 lacY1        lon::miniTn10(Tet^(R)) glnV44 galK2 rpsL20(Str^(R)) xyl-5 mtl-5        Δ(malB) zjc::Tn5(Kan^(R)) Δ(mcrC-mrr)_(HB101))    -   ER2738 (F′proA⁺B⁺ lacI^(q) Δ(lacZ)M15 zzf::Tn10(Tet^(R))/fhuA2        glnV Δ(lac-proAB) thi-1 Δ(hsdS-mcrB)5),    -   ER2925 (ara-14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA        dcm-6 hisG4 rfbD1 R(zgb210::Tn10)Tet^(S) endA1 rpsL136        dam13::Tn9 xylA-5 mtl-1 thi-1 mcrB1 hsdR2),    -   GC5™ (:F− Φ80lacZ Δ M15 Δ (lacZYA-argF)U169 endA1 recA1 relA1        gyrA96 hsdR17 (r_(k) ⁻, m_(k) ⁺) phoA supE44 thi-1λ−T1R),    -   GC10 (F− mcrA Δ(mrr-hsdRMSmcrBC) Φ80dlacZ Δ M15 Δ lacX74 endA1        recA1 Δ (ara, leu)7697 araD139 galUgalK nupG rpsL λ−T1R),    -   GENEHOGS® (FmcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1        araD139 Δ(araleu)7697 galU galK rpsL (StrR) endA1 nupG fhuA::IS2        (confers phage T1 resistance)),    -   HB101,    -   HMS174,    -   HMS174(DE3),    -   HI-CONTROL™ BL21(DE3) (F⁻ ompT gal dcm hsdS_(B)(r_(B) ⁻ m_(B) ⁻)        (DE3)/Mini-F lacI^(q1)(Gent^(r))).    -   HI-CONTROL™ 10G (F− mcrA Δ(mrr-hsdRMS-mcrBC) endA1 recA1        Φ80dlacZΔM15 ΔlacX74araD139 Δ(ara,leu)7697 galU galK rpsL nupG        λ− tonA/Mini-F lacI^(q1) (Gent^(r))),    -   HT96™ NOVABLUE (endA1 hsdR17 (r_(K12) ⁻ m_(K12) ⁺) supE44 thi-1        recA1 gyrA96 relA1 lac F′[proA⁺B⁺ lacI^(q)ZΔM15::Tn10]        (Tet^(R))),    -   IJ1126, IJ1127, INV110, JM83,    -   JM101 (F′ traD36 proA⁺B⁺ lacI^(q) Δ(lacZ)M15/Δ(lac-proAB) glnV        thi),    -   JM103, JM105, JM106, JM107, JM108,    -   JM109 (F′ traD36 proA⁺B⁺ lacI^(q) Δ(lacZ)M15/Δ(lac-proAB) glnV44        e14⁻ gyrA96 recA1 relA1 endA1 thi hsdR17),    -   JM109(DE3), JM110, JS5, KS1000 (F′ lacI^(q) lac⁺ pro⁺/ara        Δ(lac-pro) Δ(tsp)=Δ(prc)::Kan^(R) eda51::Tn10(Tet^(R))        gyrA(Nal^(R)) rpoB thi-1 argE(am)), LE392,    -   Lemo21(DE3) (fhuA2 [lon] ompT gal (λ DE3) [dcm]        ΔhsdS/pLemo(Cam^(R)) λ DE3=λ sBamHIo ΔEcoRI-B        int::(lacI::PlacUV5::T7 gene1) i21 Δnin5    -   pLemo=pACYC184-PrhaBAD-lysY),    -   LIBRARY EFFICIENCY® DH5A™ (F−φ80lacZΔM15 Δ(lacZYA-argF)U169        recA1 endA1 hsdR17(r_(k) ⁻, m_(k) ⁺) phoA supE44 thi-1 gyrA96        relA1λ−),    -   MACH1™ T1R (F− Φ80lacZΔM15 ΔlacX74 hsdR(rK−, mK+) ΔrecA1398        endA1 tonA),    -   MAX EFFICIENCY® DH10B™ (F-mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15        ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ-rpsL        nupG/pMON14272/pMON7124),    -   MC1061, MC4100, MDS™ 42(MGJ655 fhuACDB(del) endA(del)+deletion        of 699 additional genes, including all IS elements and cryptic        prophages as listed in Posfai et al. (2006) Science        (312):1044-1046), MFDpir,    -   NEB Express l^(q1)(MiniF lacI^(q) (Cam^(R))/fhuA2 [lon] ompT gal        sulA11 R(mcr-73::miniTn10-Tet^(S))2 [dcm]        R(zgb-210::Tn10-Tet^(S)) endA1 Δ(mcrC-mrr)114::IS10),    -   NEB Express, dam⁻/dcm⁻,    -   NEB 5-alpha (fhuA2 Δ(argF-lacZ)U69 phoA glnV44 Φ80Δ (lacZ)M15        gyrA96 recA1 relA1 endA thi-1 hsdR17),    -   NEB 10-beta (Δ(ara-leu) 7697 araD139 fhuA ΔlacX74 galK16 galE15        e14-φ80dlacZΔM15 recA1 relA1 endA1 nupG rpsL (Str^(R)) rph spoT1        Δ(mrr-hsdRMS-mcrBC)),    -   NiCo21(DE3) (can::CBD fhuA2 [lon] ompT gal (λ DE3) [dcm]        arnA::CBD slyD::CBD glmS6Ala ΔhsdS λ DE3=λ sBamHIo ΔEcoRI-B        int::(lacI::PlacUV5::T7 gene1) i21 Δnin5),    -   NM522 (F′ proA⁺B⁺ lacI^(q) Δ(lacZ)M15/Δ(lac-proAB) glnV thi-1        Δ(hsdS-mcrB)5),    -   NOVABLUE™ (endA1 hsdR17 (r_(K12) ⁻ m_(K12) ⁺) supE44 thi-1 recA1        gyrA96 relA1 lac F′[proA⁺B⁺ lacI^(q)ZΔM15::Tn10](Tet^(R))),    -   NovaF− (F⁻ endA1 hsdR17 (r_(K12) ⁻ m_(K12) ⁺) supE44 thi-1 recA1        gyrA96 relA1 lac),    -   NOVAXGF′ ZAPPERS™ (mcrA Δ(mcrC mrr) endA1recA1 φ80dlacZΔM15        ΔlacX74araD139 Δ(ara-leu)7697 galUgalKrpsLnupGλ⁻tonA        F′[lacI^(q)Tn10] (Tet^(R))).    -   OMNIMAX™2T1® (F′ {proAB+ lacIq lacZΔM15 Tn10(TetR) Δ(ccdAB)}        mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 Δ(lacZYA-argF)    -   U169 endA1 recA1 supE44 thi-1 gyrA96 relA1 tonA panD),    -   ONE SHOT® BL21 STAR™ (DE3) (F−ompT hsdSB (rB−, mB−) galdcmrne131        (DE3)),    -   ONESHOT® TOP10 (F− mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 Δ lacX74        recA1 araD139 Δ(araleu)7697galU galK rpsL (StrR) endA1 nupG).    -   ORIGAMI™ (Δ(ara-leu) 7697 ΔlacX74 ΔphoA PvuII phoR araD139 ahpC        galE galK rpsLF′[lac⁺ lacI^(q) pro] (DE3)gor522::Tn10 trxB        (Kan^(R), Str^(R), Tet^(R))),    -   ORAGAMI™ 2 (Δ(ara-leu) 7697 ΔlacX74 ΔphoA PvuII phoR araD139        ahpC galE galK rpsL F′[lac⁺ lacI^(q) pro] gor522::Tn10 trxB        (Str^(R), Tet^(R))),    -   OVEREXPRESS™ C41(DE3) (F− ompT hsdSB (rB− mB−) gal dcm (DE3)),    -   OVEREXPRESS™ C41(DE3)PLYSS (F− ompT hsdSB (rB− mB−) gal dcm        (DE3) pLysS (Cm^(R))),    -   OVEREXPRESS™ C43(DE3) (F− ompT hsdSB (rB− mB−) gal dcm (DE3)),    -   OVEREXPRESS™ C43(DE3)PLYSS (F− ompT hsdSB (rB− mB−) gal dcm        (DE3) pLysS (Cm^(R))),    -   POP2136/pFOS1 (F⁻ glnV44 hsdR17 endA1 thi-1 aroB mal⁻ cI857        lambdaPR),    -   PR1031 (F⁻ thr:Tn10(Tet^(R)) dnaJ259 leu fhuA2 lacZ90(oc) lacY        glnV44 thi),    -   ROSETTA™ (F⁻ompT hsdS_(B)(r_(B) ⁻ m_(B) ⁻) gal dcm pRARE        (Cam^(R))),    -   ROSETTA™ (DE3)PLYSS (F⁻ompT hsdS_(B)(r_(B) ⁻ m_(B) ⁻) gal dcm        (DE3) pLysSRARE2 (Cam^(R))),    -   ROSETTA-GAMI™ (Δ(ara-leu)7697 ΔlacX74 ΔphoA PvuII phoR araD139        ahpC galE galK rpsL F′[lac⁺ lacI^(q) pro] gor522::Tn10 trxB        pRARE2 (Cam^(R), Str^(R), Tet^(R))),    -   ROSETTA-GAMI™ (DE3)PLYSS (Δ(ara-leu)7697 ΔlacX74 ΔphoA PvuII        phoR araD139 ahpC galE galK rpsL (DE3) F′[lac⁺ lacI^(q)        pro]gor522::Tn10 trxB pLysSRARE2 (Cam^(R), Str^(R), Tet^(R))),    -   RR1, RV308, SCARABXPRESS® T7LAC (MDS™42 multiple-deletion        strain (1) with a chromosomal copy of the T7 RNA Polymerase        gene),    -   SS320 (F′[proAB+lacIqlacZΔM15 Tn10 (tet^(r))]hsdR mcrB araD139        Δ(araABC-leu)7679 ΔlacX74 galUgalK rpsL thi),    -   SHUFFLE® (F′ lac pro lacI^(q)/Δ(ara-leu)7697 araD13 fhuA2        Δ(lac)X74 Δ(phoA)PvuII phoR ahpC*galE (or U) galK        Δλatt::pNEB3-r1-cDsbC (SpecR, lacI^(q)) ΔtrxB rpsL150(StrR) Δgor        Δ(malF)3),    -   SHUFFLE® T7 (F′ lac, pro, lacI^(q)/Δ(ara-leu)7697 araD139 fhuA2        lacZ::T7 gene1 Δ(phoA)PvuII phoR ahpC*galE (or U) galK        λatt::pNEB3-r1-cDsbC (Spec^(R), lacI^(q)) ΔtrxB rpsL150(Str^(R))        Δgor Δ(malF)3),    -   SHUFFLE® T7 EXPRESS (huA2 lacZ::T7 gene1 [lon] ompT ahpC gal        λatt::pNEB3-r1-cDsbC (Spec^(R), lacI^(q)) ΔtrxB sulA11        R(mcr-73::miniTn10-Tet^(S))2 [dcm] R(zgb-210::Tn10-Tet^(S))        endA1 Δgor Δ(mcrC-mrr)114::IS10),    -   SOLR (e14-(McrA−) Δ(mcrCB-hsdSMR-mrr)171 sbcC recB recJ uvrC        umuC::Tn5 (Kan^(r)) lac gyrA96 relA1 thi-1 endA1λ^(R) [F′ proAB        lacI^(q)Z ΔM15]^(C) Su−),    -   SCS110, STBL2™ (F− endA1 gln V44 thi-1 recA1 gyrA96 relA1        Δ(lac-proAB) mcrA Δ(mcrBC-hsdRMS-mrr) λ⁻),    -   STBL3™ (F− glnV44 recA13 mcrB mrr hsdS20(rB−, mB−) ara-14 galK2        lacY1 proA2 rpsL20 xyl-5 leu mtl-1),    -   STBL4™ (endA1 glnV44 thi-1 recA1 gyrA96 relA1 Δ(lac-proAB) mcrA        Δ(mcrBC-hsdRMS-mrr) λ⁻ gal F′[proAB⁺ lacI^(q) lacZΔM15 Tn10]),    -   STELLAR™ (F−, endA1, supE44, thi-1, recA1, relA1, gyrA496, phoA,        Φ80d lacZΔ M15, Δ (lacZYA-argF) U169, Δ (mrr-hsdRMS-mcrBC),        ΔmcrA, λ−),    -   SURE (endA1 glnV44 thi-1 gyrA96 relA1 lac recB recJ sbcC        umuC::Tn5 uvrC e14-Δ(mcrCB-hsdSMR-mrr)171 F′[proAB⁺ lacI^(q)        lacZΔM15 Tn10]),    -   SURE2 (endA1 glnV44 thi-1 gyrA96 relA1 lac recB recJ sbcC        umuC::Tn5 uvrC e14-Δ(mcrCB-hsdSMR-mrr) 171 F′[proAB⁺ lacI^(q)        lacZΔM15 Tn10 Amy Cm^(R)]),    -   T7 Express Crystal (fhuA2 lacZ::T7 gene1 [lon] ompT gal sulA11        R(mcr-73::miniTn10-Tet^(S))2 [dcm] R(zgb-210::Tn10-Tet^(S))        endA1 metB1 Δ(mcrC-mrr)114::IS10),    -   T7 Express lysY/I^(q) (MiniF lvsY lacI^(q)(Cam^(R))/fhuA2        lacZ::T7 gene1 [lon] ompT gal sulA11        R(mcr-73::miniTn10-Tet^(S))2 [dcm] R(zgb-210::Tn10-Tet^(S))        endA1 Δ(mcrC-mrr) 114::IS10),    -   T7 Express lysY (MiniF lysY (Cam^(R))/fhuA2 lacZ::T7 gene1 [lon]        ompT gal sulA11 R(mcr-73::miniTn10-Tet^(S))2 [dcm]        R(zgb-210::Tn10-Tet^(S)) endA1 Δ(mcrC-mrr)114::IS10),    -   T7 Express I^(q) (MiniF lacI^(q)(Cam^(R))/fhuA2 lacZ::T7 gene1        [lon] ompT gal sulA11 R(mcr-73::miniTn10-Tet^(S))2 [dcm]        R(zgb-210::Tn10-Tet^(S)) endA1 Δ(mcrC-mrr)114::IS10).    -   T7 Express (fhuA2 lacZ::T7 gene1 [lon] ompT gal sulA11        R(mcr-73::miniTn10-Tet^(S))2 [dcm] R(zgb-210::Tn10-Tet^(S))        endA1 Δ(mcrC-mrr)114::IS10).    -   TB1 (F⁻ ara Δ(lac-proAB) [Φ80dlac Δ(lacZ)M15] rpsL(Str^(R)) thi        hsdR),    -   TG1 (F′ [traD36 proAB⁺ lacI^(q) lacZΔM15]supE thi-1 Δ(lac-proAB)        Δ(mcrB-hsdSM)5, (r_(K) ⁻m_(K) ⁻)),    -   THUNDERBOLT™ GC10 (F− mcrA Δ (mrr-hsdRMSmcrBC) Φ80dlacZ Δ M15        DlacX74 endA1recA1 Δ (ara, leu)7697 araD139 galU galK nupG rpsL        l λ-T1R),    -   UT5600 (F⁻ ara-14 leuB6 secA6 lacY1 proC14 tsx-67d(ompT-fepC)266        entA403 trpE38 rfbD1 rpsL109 xyl-5 mtl-1 thi-1),    -   VEGGIE™ BL21(DE3) (F⁻ompT hsdS_(B)(r_(B) ⁻ m_(B) ⁻) gal        dcm(DE3)), W3110 (λ857S7),    -   WM3064,    -   XL1-Blue (endA1 gyrA96(nal^(R)) thi-1 recA1 relA1 lac glnV44        F′[::Tn10 proAB⁺ lacI^(q)Δ(lacZ)laM15] hsdR17(r_(K) ⁻ r_(K) ⁺)),    -   XL1-Blue MRF′(Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44        thi-1 recA1 gyrA96 relA1 lac [F′ proAB lacI^(q)ZΔM15 Tn10        (Tet^(r))]),    -   XL2-Blue (endA1 gyrA96(nal^(R)) thi-1 recA1 relA1 lac glnV44        F′[::Tn10 proAB⁺ lacI^(q)Δ(lacZ)M15 Amy Cm^(R)] hsdR17(r_(K) ⁻        m_(K) ⁺)).    -   XL2-Blue MRF′(endA1 gyrA96(nal^(R)) thi-1 recA1 relA1 lac glnV44        e14− Δ(mcrCB-hsdSMR-mrr)171 recB recJ sbcC umuC::Tn5 uvrC        F′[::Tn10    -   proAB⁺ lacI^(q)Δ(lacZ)M15 Amy Cm^(R)]),    -   XL1-Red (F− endA1 gyrA96(nal^(R)) thi-1 relA1 lac glnV44        hsdR17(r_(K) ⁻ m_(K) ⁺) mutS mutT mutD5 Tn10),    -   XL10-Gold (endA1 glnV44 recA1 thi-1 gyrA96 relA1 lac Hte        Δ(mcrA)183 Δ(mcrCB-hsdSMIR-mrr)173 tet^(R) F′[proAB        lacI^(q)ZΔM15 Tn10(Tet^(R) Amy Cm^(R))]), and    -   XL10-Gold KanR (endA1 glnV44 recA1 thi-1 gyrA96 relA1 lac Hte        Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr) 173 tet^(R) F′[proAB        lacI^(q)ZΔM15 Tn10(Tet^(R) Amy Tn5(Kan^(R))]).

The following examples are provided to illustrate various aspects of thepresent invention, and should not be construed as limiting the inventiononly to these particularly disclosed embodiments. The materials andmethods employed in the examples below are for illustrative purposes,and are not intended to limit the practice of the present inventionthereto. Any materials and methods similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention.

Example 1 Identification of Host Cell Proteins Associated with aSpecific Product, Histidine-Tagged Green Fluorescent Protein, as aComparative Example

This comparative example demonstrates the identification of proteins ofthe 120 mM imidazole fraction (Ni(II) IMAC) and subsequent genedeletions. It demonstrates how to eliminate host cell contaminants for aspecific target recombinant product. Green Fluorescent Protein (GFPuv),extended by a histidine-rich affinity tag (His₆-GFP). His₆-GFP elutessimilarly to other histidine-tagged proteins found in the literature.While this example discloses three gene deletions that, in principle,would enhance the purity of the desired product, the knockouts of cyoA,adhP, and yfbG and their subsequent lack of expression does notfavorably impact column capacity. These three proteins are insignificantin the metalloproteome of E. coli. Thus, no changes to the separatomeare disclosed that lead to an overall increase in separation efficiency.The text of this example is an annotated version of the inventors' workdescribed in Liu et al. (2009) J. Chromatog. A 1216:2433-2438.

Strains, Plasmids, and Growth Conditions

Escherichia coli BL21 DE3 expressing GFPuv tagged with HHHHHH (His₆)(SEQ ID NO: 1) were constructed using basic molecular biologytechniques. PCR primers F(5′-GCCAAGTTGTGGCATCATCATCCGCATATGAGTAAAGGAGAAGAACTITC-3′) (SEQ ID NO:2)and R (5′-TTGGAATTCATTATTTGTAG AGCT-3′) (SEQ ID NO:3) containing HindIII and EcoRI sites (underlined correspondingly), were used to amplifyand extend GFPuv. These enzymes were used to digest the PCR fragment andthe parent plasmid. T4 DNA ligase was then used to construct a newvector that was built from the PCR-extended gene and the major part ofthe pGFPuv plasmid. Transformants were selected in LB agar containing 50μg/ml ampicillin. E. coli cells were grown in Luria-Bertani (LB)overnight and inoculated in a 2-liter flask containing 500 ml M9supplemented with 10 g/L glucose such that the initial A₆₆₀ was 0.1. Toexpress His₆-GFPuv, 4% inoculations of overnight cultures were made in500 mL LB and induced with 1 mM of IPTG after 1-2 hours. Fermentationswere carried out at 37° C. and the agitation speed of the shaker was setat 200 rpm. Cell pellets were collected by centrifugation at 5000 g andfrozen at −80 OC before cell lysis.

Sample Preparation and Chromatography

Cell pellets were suspended in 20 ml 1× native purification buffer (50mM NaH₂PO₄, pH 8.0; 500 mM NaCl) combined with 100 μl Triton X-100, 80μl 100 mM MgCl₂, 20 μl phenylmethylsulphonyl fluoride (PMSF) and 100 μl100 mg/mL lysozyme. The mixture was sonicated on ice at 4 W for 30 minusing a Vibra cell ultrasonifier (Fisher Scientific. Pittsburgh, Pa.,USA), and centrifuged at 5000 rpm for 20 min. The supernatants werecollected and passed through a 0.45 μm filter before column loading.

For experiments identifying natural contaminants or to follow theadsorption and elution of His₆-GFP, the cleared lysate was applied to 4ml ProBond nickel-chelating resin in an open column followed byequilibration using IX native purification buffer (5× nativepurification buffer, as supplied with the resin, is comprised of 250 mMNaH2PO4, pH 8.0, 2.5 M NaCl). Step elutions were carried out with nativepurification buffer with the following imidazole concentrations: 60 mM,80 mM, 100 mM, 120 mM, and 200 mM. This was followed by a 500 mM EDTAelution. The elution volumes for each step were 24 ml, or 6 columnvolumes (CVs), and applied at an approximate flow rate of 0.5 ml/min.Fractions were collected and measured for protein concentration with aBCA Protein Assay Kit (Pierce, Rockford, Ill. USA) and/or assayed forGFPuv in triplicate with a Tecan Infinite M200 96-well plate reader withexcitation/emission spectra set to 395/509 nm.

SDS-PAGE and Mass Spectrometry

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) wasperformed for 6 hours at 100 V. Gels were stained with Coomassie Blue.The Genomics and Proteomics Core Laboratories at the University ofPittsburgh performed the protein identification. To account for theexperimental accuracy of the measurement, three spots were excised fromeach band and each digested with trypsin. Peptides were separated byliquid chromatography (LC), then identified by tandem mass spectrometry(MS/MS) fragmented by collision-induced dissociation. MASCOT v2.1(Matrix Science, Boston Mass. USA) was used to match LC/MS data with E.coli proteins. For positive identification, spectral data from each ofthe three spots matched.

Functional Prediction of Identified Proteins in 120 mM Elution Fraction

Functional classification of all identified proteins was based on theProfiling of Escherichia coli chromosome (PEC) database (Hashimoto etal. (2005) Molecular Microbiology 55; 137-149).

Construction of Knockout Mutants

All the knockout mutants of this Example were generated with the samedeletion system according to the manual accompanying the Quick and EasyE. coli Gene Deletion Kit (Gene Bridges, Heidelberg, Germany). This kituses plasmid pRedET to facilitate homologous recombination events.During the progression of the work, a triple mutant of BL21(ΔcyoAΔyfbGΔadhP) was constructed through a series operation consistingof recombination, selection with kanamycin, confirmation, and removal ofthe selection marker using flipase recognition site (FRT flankedkanamycin gene).

Southern Blot Analysis

DNA probes used for Southern hybridization were prepared fromPCR-amplified fragments. Probes were labeled according to the manual ofAmersham Gene Images Random Prime Labeling Kit (GE Healthcare). GenomicDNA was isolated from knockout mutants using standard protocols. DNAsamples were digested with Bam HI, separated by electrophoresis on 1%agarose gels, transferred to Amersham Hybond-N+ membranes (GEHealthcare), and then baked at 80° C. for 2 hours. The probes werehybridized to these blots and detected according to the protocol of theGene Images ECL Detection Kit (GE Healthcare).

SDS-PAGE Evaluation of CyoA, YfbG and AdhP Knockout in Mutant Strains

Cell preparations of BL21, mutants, and chromatography fractions wereevaluated by SDS-PAGE. Approximately 15 μg sample/well were loaded intoa 120% acrylamide gel.

Identification of Knockout Candidates and Confirmation of their Deletion

A total extract of E. coli protein was loaded to the ProBondnickel-chelating column using 1× native purification buffer (5× nativepurification buffer, as supplied with the resin, is comprised of 250 mMNaH2PO4, pH 8.0, 2.5 M NaCl). Step elutions were carried out with nativepurification buffer with the following imidazole concentrations: 60 mM,80 mM, 100 mM, 120 mM, and 200 mM. FIG. 3 shows the proteinconcentrations in each fraction normalized to the total protein used forcolumn loading. The bar graph indicates order of magnitude changes inthe total protein encountered with each imidazole challenge. Note thatthe elution fraction containing the 120 mM imidazole fraction containedthe least amount of protein. Coincidentally, this fraction that containslow host cell protein is also the fraction where His₆-GFPuv elutes.

SDS-PAGE and LC-MS/MS were used to identify the cellular proteinspresent in the concentrated sample of pooled 120 mM imidazole elutionfractions. A total of 18 proteins were identified (Table 2), with cyoA,yfbG, and adhP selected for deletion due to lack of essentiality.Southern blot analysis and gel electrophoresis indicated lack ofexpression of the three gene products cyoA, yfbG, and adhP. FIG. 4 showsthis confirmation due to lack of spots associated with positivehybridization and bands of the molecular weights of these products,respectively.

TABLE 2 Proteins eluted at 120 mM from a Ni(II)-NTA column dnaK yfbGadhP cyoA rplB slyD nagD ahpC rpsG rplO rpsE rplM Fur Hypotheticalprotein ECs2542 rplJ rpsL Hns rplL

These results demonstrate that it is possible to apply a limited set ofdata and to produce a knockout strain that might be capable of enhancingthe purity of a recombinant peptide, polypeptide, or protein. It is usedas a comparative example to illustrate the lack of a rigorousmethodology to identify specific changes to the host cell that lead toan altered separatome capable of broadly improving separationefficiency, and column capacity in particular, regardless of desiredrecombinant product. By focusing on the contaminants of a singlespecific his-tagged protein in a particular column fraction. Liu et al.(2009) J. Chromatog. A 1216:2433-2438 fails to even consider theprinciple of prioritizing host cell contaminant proteins that, ifdeleted, modified, or inhibited, would significantly improve columncapacity and/or selectivity (“chromatographic separation efficiency”)for a wide variety of different recombinant peptides, polypeptides, andproteins as disclosed and claimed herein.

Example 2 Construction of an Ion Exchange Separatome of E. coli and itsUse to Design and Build Novel Host Strains for a Common ChromatographyResin

This example describes the process by which a separatome is constructedfor a chromatography resin and subsequently used to guide modificationsto E. coli to increase chromatographic efficiency. It begins bydescribing how data are acquired by fractionating an extract derivedfrom fed batch growth over a DEAE ion exchange bed, and continues byconstructing the separatome—a data structure that includes informationon the genes responsible for identified proteins coupled to aquantitative scoring to rank order molecular biology efforts that leadto a reduced separatome. Finally, construction of example strains isdescribed, concluding with information regarding high priority strainmodifications necessary for significant gains in separation efficiencythrough their deletion, modification, or inhibition.

Section I. Cloning Strains and Vectors

E. coli strain MG1655 (K-12 derivative) was selected as the base strainfor cell line modification because of its widespread use and lack ofcommercial license. Its genotype is F lambda⁻ rph⁻¹, meaning that itlacks an F pilus, the phage lambda, and has a15 codon frame-shift asresult of the rph 1 bp deletion (Yale University. E. coli Genetic StockCenter Database. 2013). This frame-shift interrupts the pyrE gene andreduces pyrimidine levels (Jensen et al. (1993) Journal of Bacteriology175(11):3401-7).

Plasmid pKD46 was used as part of the λ-red recombination system. Thisplasmid is ampicillin resistant and replication is temperaturesensitive. For plasmid maintenance, growth is at 30° C. and the plasmidcan be removed by growth at 37° C. without antibiotic pressure. Theplasmid encodes for lambda Red genes exo, bet, and gam, and includes anarabinose-inducible promoter for expression (Datsenko et al. (2000) PNAS97(12):6640-5). The plasmid was provided in conjunction with MG1655 fromthe Yale E. Coli Genetic Stock Center (New Haven, Conn.).

Expression Strains and Vectors

E. coli strain BL21 (DE3) was used for initial cell culture and celllysate preparation. Its genotype is F− ompT hsdSB(rB−, mB−) gal dcm(DE3). The strain and genotype was provided by Novagen(EMD-Millipore/Merck). The cell line was transformed with a recombinantpGEX plasmid provided by Dr. Joshua Sakon (Department of Chemistry,University of Arkansas). This plasmid, pCHC305, contains the geneticinformation for the recombinant fusion protein,glutathione-S-transferase—parathyroid hormone—collagen binding domain(GST-PTH-CBD, 383 amino acids).

Storage Strains and Vectors

For storage of DNA constructs, E. coli strain DH5α was selected. Itsgenotype is F−, Δ(argF-lac)169 φ80dlacZS8(M15) ΔphoA glnV44(AS)8λ−deoR481 rfbC gyrA96(NalR)1 recA1 endA1 thiE1 hsdR17. DH5 is anon-mutagenized derivative of DH1, which transforms more efficiently dueto a deoR mutation. The recA mutation eliminates homologousrecombination and minimizes undesired modification to stored plasmids.

pUC19 was used as a DNA storage vector. It is a high copy number plasmidthat carriers ampicillin resistance. This plasmid was provided inconjunction with DH5α from the Yale E. Coli Genetic Stock Center (NewHaven, Conn.).

Liquid Growth Media

M9 medium was used where a minimal defined medium was required. M9Medium was made in 3 separate stock solutions: glucose solution (500g/L), trace elements (2.8 g of FeSo4-7H2O, 2 g of MnCl2-4H2O, 2.8 g ofCaCl2-7H2O, 1.5 g of CaCl2-2H2O, 0.2 g CuCl2-2H2O, 0.3 g of ZnSO4-7H2O),and 5×M9 (75 g of K2HPO4, 37.5 g of KH2PO4, 10 g of citric acid, 12.5 gof (NH4)2SO4, 10 g of MgSO4-7H2O). Each of these components must beautoclaved individually to minimize salt precipitation. To prepare 1 Lof M9, 20 ml of the glucose solution is mixed with 1 ml trace elementsolution, 200 ml of 5×M9, and enough water to bring the final volume upto 1 L (approximately 780 ml).

Where rich medium was required, Luria-Burtani (LB) Medium was used. LBpowder was purchased from Difco and was prepared per the manufacturer'sinstructions: 20 g LB powder per 1 L of milliQ water.

Solid Growth Media

Solid M9 medium was prepared as previously described for liquid M9 withthe addition of agar to the water and concentrated M9 solution prior toautoclaving. To prepare 500 ml of M9 agar, 7.5 g agar, 100 ml of 5×M9solution, and 300 ml of water are mixed and autoclaved. Added to this is10 ml sterile glucose solution (500 g/L), 500 μl trace elements, andenough sterile water to bring the final volume up to 500 ml. The othersolid medium used was LB agar, which was prepared the same as the LBliquid medium described earlier plus the addition of 7.5 g agar perliter.

Fed-Batch Cultivation

Fed-batch cultivation was used to prepare the cell lysate for use indownstream protein purification and identification of natively expressedproteins. The cell line used was BL21(DE3):pCHC305. To beginfermentation, a single colony was isolated from a LB ampicillin agarplate and transferred to a 5 ml culture tube containing liquid LB plus150 μg/ml ampicillin. This culture tube was allowed to incubateovernight at 37° C. After overnight growth, the 5 ml culture tube issupplemented with 100 ml of M9 with ampicillin and allowed to grow at37° C. for six to eight hours. This 100 ml culture is then centrifugedat 4750 rpm for 25 minutes (Beckman Coulter Allegra) and re-suspended in50 ml of fresh M9 medium with 150 μg/ml ampicillin. This culture wasused as the inoculant for the fed-batch growth. The 3-liter Applikonbioreactor (Foster City, Calif.) contained 1 liter of M9 plus 150 μg/mlampicillin and 1 ml silicone anti-foam.

The Applikon unit was equipped with BioXpert Advisory software fromApplikon, an Applisense pH probe, and a dissolved oxygen probe. Tomaintain proper dissolved oxygen, the reactor was supplemented with pureoxygen provided by a compressed gas cylinder with a controllable flowrate. To insure effective gas dispersal, the culture was initiallystirred at 750 rpm and was later increased to 1000 rpm based on celldensity. Adjustments in oxygen delivery were made as necessary duringthe process to ensure that the dissolved oxygen concentration did notdrop below 35%. The pH was maintained at approximately 6.8 (with a rangeof 6.75 to 7) during the cultivation by adding 7M NH₄OH as needed.Temperature was maintained at 37° C. using a heating jacket and coolingloop. Optical densities were monitored using a Bugeye optical densityprobe (BugLab, Foster City, Calif.) and a DU800 Beckman Coulterspectrophotometer (Brea, Calif.). A linear correlation for the Bugeyeresponse to the actual optical density (OD) as measured by thespectrophotometer was determined for each individual experiment.

The fed-batch fermentation process has two phases, a batch phase and afeeding phase. In the batch phase, the culture uses only the carbonsources provided in the media at the start of the cultivation and nonutrients are fed to the reactor. This phase lasted approximately 7-8hours, depending on the lag phase of the culture and how rapidly theculture grew on the initial carbon substrate. The shift from batch phaseto feeding phase can be determined by two indicators, a rise in pH and asharp decline in oxygen concentration, which indicate that the initialcarbon substrate has been depleted. In the fed-batch experiments, thesetwo events occur simultaneously and are displayed by the Applikonsoftware. The feeding profile used for fermentation experiments is basedon that of a collaborator (McKinzie Fruchtl) and was originally proposedby Korz et al. (1995) Journal of Biotechnology 39(1):59-65 and Lee etal. (1996) Trends in Biotechnology 14(3):98-105. A feeding profile wasprogrammed into the Applikon software that mimics the exponential feedbased on substrate concentrations.

An exponential fed-batch fermentation method commonly used topre-determine the amount of glucose that should be fed into the reactorto achieve a certain growth rate was proposed by Korz et al. and Lee etal., supra:

$\begin{matrix}{{M_{s}(t)} = {{{F(t)}{S_{F}(t)}} = {{\left( {\frac{\mu}{Y_{X\text{/}S}} + m} \right){X(t)}{V(t)}} = {\left( {\frac{\mu}{Y_{X\text{/}S}} + m} \right){X\left( t_{F} \right)}{V\left( t_{F} \right)}\exp^{\mu {({t - t_{F}})}}}}}} & {{Equation}\mspace{14mu} 4}\end{matrix}$

where M_(S) is the mass flow rate (g/h) of the substrate, F is thefeeding rate (l/h), S_(F) is the concentration of the substrate in thefeed (g/l), μ is the specific growth rate (l/h). Y_(X/S) represents thebiomass on substrate yield coefficient (g/g), m is the maintenancecoefficient (g/g h), and X and V represent the biomass concentration(g/l) and cultivation volume (l), respectively. The yield coefficientfor E. coli on glucose is generally taken to be 0.5 g/g (Korz et al.,supra; Shiloach et al. (2005) Biotechnology Advances 23(5):345-57). Themaintenance coefficient is often 0.025 g/g h (Korz D J et al., supra).This equation has been widely adapted for fed-batch fermentationprocesses, as exponential feeding allows cells to grow at a constantrate (Kim et al. (2004) 26(3):147-50).

During fed-batch fermentation, the cells were left un-induced toprevent/minimize the addition of the recombinant protein to the nativeprotein pool. This strategy provided the stress associated with plasmidmaintenance common to all bacterial fermentations where the gene for thetarget peptide, polypeptide, or protein is housed on a plasmid.Furthermore, this strategy permits the derivation of a separatome thatis not biased by large amounts of target peptide, polypeptide, orprotein, attesting to the universal nature of the approach. Thefermentation was allowed to grow for a total of 24 hours frominoculation to harvest. At the end of the fermentation process, cellswere harvested from the reactor by pumping the reactor contents intocentrifuge bottles. The reactor contents were then centrifuged at12,000×g for 30 minutes at 5° C. (Beckman Coulter Avanti, JLA-10.500fixed angle rotor) to separate the cell pellet from the media. Thepellet was separated into four 50 ml conical bottom tubes for storage at−20° C.

Lysate Preparation

One of the 50 ml pellets (58.9 g) was re-suspended in 150 ml of 25 mMTris buffer, pH 7. To enable cell lysis, 2 mg/ml lysozyme were added tothe mixture. In addition, 1 mM phenylmethylsulphonyl fluoride (PMSF), 20μg/ml aprotinin, and 1 mM ethylenediamine-tetraacetic acid (EDTA) wasadded to minimize protein degradation. The mixture was then incubated onice with stirring for 30 minutes to lyse the cells. The mixture was thencentrifuged at 50,000×g (Beckman Coulter Avanti, JA-25.50 fixed-anglerotor) for 30 minutes at 5° C. to separate the proteins from the celldebris.

The proteins in the supernatant were carefully pipetted out of thecentrifuge tubes, to minimize contaminants from the insoluble fraction,and were clarified by syringe filtration through 0.45 μm celluloseacetate. Lastly, the total protein concentration of the cell lysate wasdetermined by using a Bio-Rad DC Protein Assay which is a detergentcompatible colorimetric assay that is read by spectrophotometer at 750nm (Beckman Coulter DU 800 HP). Bovine serum albumin standards were usedto determine the baseline correlation between protein concentration andabsorbance at 750 nm.

Fast Protein Liquid Chromatography

Fast protein liquid chromatography (FPLC) was used to separate thenatively expressed proteins into groups based on the salt concentrationat which they elute, which correlates to their surface charge. Thechromatography was performed using an Amersham ÄkTA FPLC. The systemconsists of dual syringe pumps (P-920), gradient mixer, a monitor(UPC-900) for UV (280 nm), pH and conductivity, a fraction collector(Frac-900) and UNICORN® V3.21 data collection and archive software.

Resin

For the initial separatome database development, diethylaminoethylcellulose (DEAE) was selected as the ion exchange (IEX) resin due to itsprevalence of use in industrial manufacturing. Specifically, the columnused was a 1 ml HiTrap DEAE FF from GE Healthcare. DEAE is a weak anionexchanger, meaning that it is a positively charged matrix with a narrowworking pH of 2-9 (GE Healthcare. Instructions 71-5017-51 AG HiTrap ionexchange columns, 1-24).

Buffer Composition

25 mM Tris buffer, pH 7, was selected for all of the FPLC purificationsteps. The loading buffer contained 10 mM NaCl to minimize non-specificbinding (Buffer A). The elution buffer contained 1M NaCl, which issufficient to desorb bound proteins (Buffer B).

Column Loading Conditions

Prior to loading the column, the system was washed with buffer A untilequilibrium was achieved (roughly 10 ml). At this point, all systemmonitors were base-lined. The column was loaded at 10% breakthrough asper industry standard. The amount of total lysate to be applied to thecolumn to achieve this breakthrough was determined as follows. Accordingto GE Healthcare, the dynamic binding capacity (DBC) of HiTrap DEAE FFis 110 mg HSA (human serum albumin)/ml solvent (resin). This numbergives the amount of protein that can be bound per milliliter of resin.The next step was to determine what percentage of the native proteinsbound to the DEAE resin at pH 7. To do this, 5 ml of lysate was loadedon the column and washed with 10 ml of buffer A. The flow-through wascollected in a single fraction. The column was then washed with thebuffer B and the resulting flow-through was collected. Both fractionswere then analyzed for their total protein concentration using thepreviously mentioned Bio-Rad assay. The amount of lysate (ml) to loadonto the column was determined by the following equation:

$\begin{matrix}{{{lysate}({ml})} = \frac{{DBC}*\left( {1 + \%_{BT}} \right)*V_{c}}{\%_{bound}*C_{l}}} & {{Equation}\mspace{14mu} 5}\end{matrix}$

where DBC is the dynamic binding capacity of the resin (mg/ml), %_(BT)is the desired percent breakthrough, V_(c) is the volume of the column(ml), %_(bound) is the percent of the total lysate that binds to theresin, and C₁ is the protein concentration of the lysate (mg/ml).

The column was loaded at 1 ml/min and then washed with 10 column volumes(CV) of buffer A to remove any unbound proteins. The unbound fractionwas collected for later analysis.

Column Elution Conditions

To identify where the bulk of the bound proteins eluted, the proteinswere desorbed through roughly 100 mM salt steps from 10 mM to 1M. Thisprocess allows for the identification of the priority salt fractionsthat need to be spaced out into smaller steps for later analysis.

TABLE 3 10% Elution Windows Step NaCl Length Step # % B (mM) (CV) wash0% 10 10 1 10% 109 5 2 20% 208 5 3 30% 307 5 4 40% 406 5 5 50% 505 5 660% 604 5 7 70% 703 5 8 80% 802 5 9 90% 901 5 10  100% 1000 5 clean 100%1000 5

The flow rate was maintained at 1 ml/min and the pressure limit was setto 0.5 MPa for the duration of the experiment. During elution, allfractions were collected and immediately stored at 2° C. to reduceprotein degradation. After all of the proteins have been desorbed in the1000 mM step, the fraction collector is stopped and the column iscleaned with buffer B to ensure all proteins have been desorbed andwashed out of the column. The column is then washed with sufficientbuffer A to re-equilibrate the column.

For finer focusing on the primary elution windows, smaller 5% steps areused (Table 4). In this instance, the focus was on the 10 mM to 500 mMwindow.

TABLE 4 5% Elution Windows Step NaCl Length Step # % B (mM) (CV) wash 0%10 20 1 5% 59.5 15 2 10% 109 15 3 15% 158.5 15 4 20% 208 15 5 25% 257.515 6 30% 307 15 7 35% 356.5 15 8 40% 406 15 9 45% 455.5 15 10  50% 50515 wash 100% 1000 20

Analytical Assays

Sample Processing

Prior to the samples undergoing further analysis, they were concentratedusing a GE Lifesciences Vivaspin 20 (5,000 MWCO). This reduced the 20 mlfractions to 2 ml total volume. This was split into two 1 ml samples,one was sent for LC-MSIMS, and the other was kept for SDS-PAGE.

Protein Gels—SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) wasused to observe the approximate number of proteins in each FPLC saltfraction and their molecular weight. Prior to SDS-PAGE, the samples weredesalted by buffer exchange. To do this, the previously mentioned 1 mlsample of the desired fraction was concentrated in a GE LifesciencesVivaspin 2 (5,000 MWCO) and re-suspended in 25 mM Tris buffer, pH 7. Theconcentration and re-suspension process was repeated two more times toensure all salt had been removed. After the last concentration step, thesample was left in its concentrated form to be loaded onto the SDS-PAGE.

A Bio-Rad Protean II system was used for the electrophoresis with SDSbuffer. The SDS buffer is made as a 10× stock, where the 1× runningbuffer is 25 mM Tris, 192 mM glycine, and 0.1% SDS at a pH of 8.6. Forvisualization of the chromatography samples, a 12.5% gel was used. Thesamples are mixed 5:1 with a 5× loading dye.

Electrophoresis was carried out at 100V until the sample was through thestacking gel, then increased to 140V. Average run time was around 1hour. Gels were stained using a Coomassie Blue stain (40% methanol, 10%acetic acid and 0.5% Coomassie blue) for 3 hours and then de-stainedwith a 10% acetic acid and 40% methanol solution. Gel images werecaptured by scanning on a computer flatbed scanner.

Liquid Chromatography Mass Spectroscopy (LC-MS/MS)

Samples of each FPLC salt fraction were sent to Bioproximity (Chantilly,Va.) for protein identification via liquid chromatography massspectroscopy (LC-MS/MS). The protocol for the LC-MS/MS was provided byBioproximity as follows.

Protein Denaturation and Digestion

Prior to digestion, proteins were prepared using the filter-assistedsample preparation (FASP) method (Wisniewski et al. (2009) 6(5):359-62).Next, the sample was mixed with 8 M urea, 10 mM dithiothreitol (DTT), 50mM Tris-HCl at pH 7.6 and sonicated briefly. Samples were thenconcentrated in a Millipore Amicon Ultra (30,000 MWCO) device andcentrifuged at 13,000×g for 30 min. The remaining sample was bufferexchanged with 6 M urea, 100 mM Tris-HCl at pH 7.6, then alkylated with55 mM iodoacetamide. Concentrations were measured using a Qubitfluorometer (Invitrogen). The urea concentration was reduced to 2 M,trypsin was added at a 1:40 enzyme to substrate ratio, and the sampleincubated overnight on a Thermomixer (Eppendorf) at 37 C. The Amicon wasthen centrifuged and the filtrate collected.

Peptide Desalting

Digested peptides were desalted using C18 stop-and-go extraction (STAGE)tips (Rappsilber et al. (2003) Analytical Chemistry, American ChemicalSociety 75(3):663-70). For each sample, the C18 STAGE tip was brieflyactivated with methanol, and then conditioned with 60% acetonitrile and0.5% acetic acid, followed by 2% acetonitrile and 0.5% acetic acid.Samples were loaded onto the tips and desalted with 0.5% acetic acid.Peptides were eluted with a 60%/o acetonitrile, 0.5% acetic acidsolution and dried in a vacuum centrifuge (Thermo Savant).

Liquid Chromatography-Tandem Mass Spectrometry

Peptides were analyzed by LC-MS/MS. LC was performed on an Easy-nanoLCII HPLC system (Thermo). Mobile phase A was 94.5% MilliQ water, 5%acetonitrile, 0.5% acetic acid. Mobile phase B was 80% acetonitrile,19.5% MilliQ water, 0.5% acetic acid. The 120 min LC gradient ran from2% B to 50% B over 90 min, with the remaining time used for sampleloading and column regeneration. Samples were loaded to a 2 cm×100 umI.D. trap column positioned on an actuated valve (Rheodyne). The columnwas 13 cm×100 μm I.D. fused silica with a pulled tip emitter. Both trapand analytical columns were packed with 3.5 μm C 18 resin (Magic C18-AQ, Michrom). The LC was interfaced to a dual pressure linear iontrap mass spectrometer (LTQ Velos, Thermo Fisher) via nano-electrosprayionization. An electrospray voltage of 2.4 kV was applied to apre-column tee. The mass spectrometer was programmed to acquire, bydata-dependent acquisition, tandem mass spectra from the top 15 ions inthe full scan from 400-1400 m/z. Dynamic exclusion was set to 30seconds.

Data Processing and Library Searching

Mass spectrometer RAW data files were converted to MGF (Mascot genericformat) using msconvert (Kessner et al. (2008) Bioinformatics24(21):2534-6). Detailed search parameters are printed in the searchoutput XML (extensible markup language) files. All searches requiredstrict cryptic cleavage, up to three missed cleavages, fixedmodification of cysteine alkylation, variable modification of methionineoxidation and expectation value scores of 0.01 or lower. Searches usedthe sequence libraries: UniProt Escherichia coli (strain B/BL21-DE3, TheUniProt Consortium (2012) Nucleic Acids Research 40 (Databaseissue):D71-5), the common Repository of Adventitious Proteins (cRAP)(The Global Proteome Machine, Common Repository of AdventitiousProteins, 2012 Jan. 1) and the given sequence for plasmid productGST-PTH-CBD. MGF files were searched using X!!Tandem (Craig et al.(2004) Bioinformatics 20(9):1466-7) using both the native and k-score(MacLean et al. (2006) Bioinformatics 22(22):2830-2) scoring algorithmsand by the Open Mass Spectrometry Search Algorithm (OMSSA) (Geer et al.Journal of Proteome Research 3(5):958-64). All searches were performedon Amazon Web Services-based cluster compute instances using theProteome Cluster interface. XML output files were parsed andnon-redundant protein sets determined using MassSieve. Proteins wererequired to have two or more unique peptides across the analyzed sampleswith E-value scores of 0.01 or less, 0.001 for X!Hunter and proteinE-value scores of 0.0001 or less.

Protein Quantitation

Proteins were quantified the spectral counting method (Liu et al. (2004)Analytical Chemistry 76(14):4193-201). This results in a hit count,which is approximate of protein concentration in the sample.

Database Construction

Compilation of Data

The received LC-MS/MS data was imported into Microsoft Access 2010 fordata management. The EcoGene's EcoTools Database Table Download (Rudd KE, Database Table Download|EcoGene 3.0. Department of Biochemistry andMolecular Biology R-629, University of Miami Miller School of Medicine;2012) was used to supplement the received LC-MS/MS data with additionalgenomic and proteomic data. The data added were: the protein length (inamino acids), direction of replication (clockwise or counterclockwise),left end position of the gene (in base pairs), right end position of thegene (in base pairs), molecular weight of the protein, common gene name,synonym gene name, protein name, protein function, description, GenBankGI ID (Benson et al. (2013) GenBank. Nucleic acids Research 41 (Databaseissue):D36-42) and UniProtKBiSwiss-Prot ID (The Uniprot Consortium(2012) Nucleic acids Research 40 (Database issue):D71-5). The EcoGeneCross Reference Mapping and Download tool was used to Bnum (Blattnernumber) (Blattner et al. (1997) Science 277(5331):1453-62). MicrosoftAccess was used to build relationships between the various datasets thatallowed for searches across the compiled database.

Gene essentiality data were retrieved from Gerdes et al. (2003) J.Bacteriol. 185(19):5673-84) which compiles gene essentiality from theirown research as well as the Profiling of E. coli Chromosome (PEC)database (Hashimoto et al. (2005) Molecular Microbiology 55(1): 137-49;Kato and Hashimoto (2007) Molecular Systems Biology 3(132):132; and KangY et al. (2004) J. Bacteriol. 186(15):4921-30).

All of the compiled data represent a portion of the DEAE separatomedatabase and are the foundation for future work in this area. Thisexample describes a DEAE separatome unique to choice of resin andloading condition (pH and NaCl concentration). Other combinations ofresins and loading conditions can be used to define additionalseparatomes, the compilation of which is of commercial significance.

Data Manipulation

Proteins within a separatome can reduce chromatographic efficiency. Inorder to determine the priority of genes to be deleted, each gene wasgiven a score for each elution window (shown in Table 4). Thiscriterion, or importance score, was defined by:

$\begin{matrix}{{importance}_{i} = {\sum_{j}\; \left\lbrack {{b_{1}\left( \frac{y_{cj}}{y_{\max}} \right)}\left( \frac{h_{i,j}}{h_{i,{total}}} \right)\left( \frac{h_{i,j}}{h_{j,{total}}} \right)\left( \frac{{MW}_{i}}{{MW}_{ref}} \right)^{\alpha}} \right\rbrack_{i}}} & {{Equation}\mspace{14mu} 3}\end{matrix}$

with the following definitions: b1=scaling parameter; y_(cj) andy_(max)=concentration of mobile phase eluent in fraction (j) and maximumvalue, respectively; and h_(i,j) and h_(i,total)=the amount of protein(i) in fraction (j) and total bound protein (i), respectively; andh_(j,total)=total amount of protein in fraction (j); MWi=molecularweight of protein (i); MWref=molecular weight of a reference proteinwithin the separatome; α=steric factor; and i=protein.

We chose to define the Importance Score (IS) in this fashion because theempirically derived equation captures the characteristics of bothbinding and elution data without solving numerical models ofmulti-component liquid chromatography to define the association anddissociation rate constants.

In the function given, the score can range from 1 (high negative impacton column capacity) to 0 (low or no impact on column capacity). Thesummation ranges over the desired elution windows (j) and can beadjusted to cover all of the windows, or target a select few. The firstratio accounts for adsorption strength with y_(cj) being theconcentration of the elution solvent (in the case of ion exchange, thisis NaCl) and y_(max) being the maximum solvent concentration. The secondratio accounts for adsorption specificity with h_(i,j) being the proteinconcentration in the window, over the total protein concentration in allwindows (h_(i,total)). For proteins that elute in only one window, thisvalue will be 1, where proteins that elute in multiple windows will havea lower ratio. The third ratio describes the relative amount a proteinhas in a given fraction, and the forth ratio accounts for thepossibility of steric hindrance.

A protein that remains bound and requires stringent conditions forelution exhibits a ratio

$\left( \frac{y_{cj}}{y_{\max}} \right)$

close to, or equal to, unity, whereas a protein that emerges as a tightpeak has a

$\left( \frac{h_{i,j}}{h_{i,{total}}} \right)$

ratio close to unity. Finally, a

$\left( \frac{h_{i,j}}{h_{j,{total}}} \right)$

ratio is close to unity if it constitutes the majority of fraction orelution window(j).

Molecular weight is included in the fourth ratio of the IS since itplays a role when the column is under fully loaded or breakthroughloading conditions. This ratio is raised to α, where the α term accountsfor column saturation, wherein when the column is fully saturated α=1and when the column is unsaturated α<1. This causes the MW term to bedropped in cases where the column is not fully saturated, and thusmolecular weight, or the approximate size, of the protein does notfactor into overall column capacity.

The IS equation is then used to analyze a separatome database. When usedto analyze the aforementioned DEAE separatome database for example, generpoB is present in all fractions of the gradient and has a h_(i,total)value of 1120, with ratio values of

$\left( \frac{h_{i,j}}{h_{i,{total}}} \right)$

ranging from 8.98×10³ to 0.52. For proteins that elute in only one ortwo windows, for example gene rsd, this ratio will be close to 1. Theprotein resulting from gene skp only elutes in one window, and thus hasa h_(i,total) value of 17 and a single non-zero

$\left( \frac{h_{i,j}}{h_{i,{total}}} \right)$

ratio value of 1. For gene rpoB in the 100 mM elution window, the ratioof

$\left( \frac{h_{i,j}}{h_{j,{total}}} \right)$

is 1.8×10⁻³, indicating that the gene product of rpoB is a minimalcontributor to contamination in that elution window. The fourth ratioaccounts for steric hindrance as a function of molecular weight. Again,the gene product of rpoB has a molecular weight of 150 kDa. Thismolecular weight is divided by the protein with the largest molecularweight, mukB at 170 kDa, to yield a ratio of 0.88. In this case, the αterm is 1 because the column was loaded to 10% breakthrough. Finally theb₁ term for rpoB is zero, thus forcing the IS to zero, because gene rpoBis considered essential for cellular growth. The b₁ term for skp is 1because the gene product is considered unessential. This demonstratesthat while the importance of HCP contaminants can be ranked via theimportance equation, their deletion, modification, inhibition, etc., toimprove chromatographic separation capacity may not be feasible due totheir essentiality for acceptable cell growth, viability, etc., infermentation. As discussed earlier in connection with the definition of“essential genes”, however, there are potential ways to circumvent thisproblem.

Table 5 presents the calculations for the aforementioned in tabularformat, describing the mathematics associated with rpoB and skp for the100 mM NaCl fraction (j=100); the math was repeated for all theremaining salt fractions (j=50, 150, 200, 250, 300, 350, 400, 450, 500,1000) and summed to determine the total importance score and thusdetermine the ranking in terms of the gene's negative impact on totalcolumn capacity. A high importance score indicates a large negativeimpact on chromatographic separation efficiency.

TABLE 5   Gene Name NaCl Fraction (j)     b₁$\left( \frac{y_{c,_{j}}}{y_{\max}} \right)$$\left( \frac{h_{i,_{j}}}{h_{i,{total}}} \right)$$\left( \frac{h_{i,_{j}}}{h_{i,{total}}} \right)$$\left( \frac{{MW}_{i}}{{MW}_{ref}} \right)$     α     IS₁₀₀ rpoB 100 0100/1000 12/1120 12/20556 150632/170230 1 0 skp 100 1 100/1000 17/17 17/20556  17688/170230 1 8.6E-06

Once the final IS is determined, rpoB tied for last place in the rankingdue to the b₁ of zero (due to gene essentiality, as disclosed by Gerdeset al. (2003) J. Bacteriol. 185(19):5673-84), and skp is ranked 333.From the IS, it can be determined that while rpoB has a large impact oncolumn capacity, it cannot be deleted due to its impact on cellviability and skp has such little impact that it does not meritdeletion.

In contrast to literature such as Liu et al. (2009) J. Chromatog. A1216:2433-2438, Bartlow et al. (2011) Protein Expression andPurification 78:216-224, Bartlow et al. (2012) American Institute ofChemical Engineers Biotechnol, Prog. 28:137-145, and Campbell et al.U.S. Pat. No. 8,178,339, which might suggest the removal of skp if atarget recombinant protein would co-elute in the 100 mM fraction, themethodology presented in this example demonstrates that the potentialcolumn capacity improvement from this deletion would result in anegligible column capacity improvement of approximately less than 0.01%.This demonstrates that the present separatome concept employing theimportance equation provides a novel quantitative and rational means ofidentifying and ranking host cell proteins that negatively impactchromatographic separation capacity, and therefore chromatographicselectivity and purity of the final recovered target product. Onceidentified and ranked in this way, such host cell chromatographynuisance proteins can be deleted, modified, or inhibited to produceoptimized host cells for recombinant expression of a broad spectrum oftarget peptides, polypeptides, and proteins, where such cells stillmaintain good (or possibly even improved) fermentation characteristicssuch as growth rates, viability, protein expression, etc.

The second and third equations define how much capacity is recoveredwhen the protein is removed, and the overall capacity recovery as onemodifies, deletes, or inhibits n genes, respectively

$\begin{matrix}{{{{recovery}\mspace{14mu} {potential}_{i}} = {h_{i,{total}}\text{/}h_{{total},{ms}}}}{and}} & {{Equation}\mspace{14mu} 1} \\{{{capacity}\mspace{14mu} {recovery}} = {100\% \mspace{14mu} x{\sum\limits_{i = 1}^{n}\; {{recovery}\mspace{14mu} {potential}_{i}}}}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

Homologous Recombination

Flexible Recombineering Using Integration of thyA (FRUIT) as describedby Stringer et al., FRUIT, a Scar-Free System for Targeted ChromosomalMutagenesis, Epitope Tagging, and Promoter Replacement in Escherichiacoli and Salmonella enterica. PoS one. 2012 January; 7(9):e44841, amodification of the Datsenko λ-Red homologous recombination system, wasused to delete the targeted genes from the genome of E. coli strainMG1655. This is a new system, which utilizes the gene thyA as a growthoriented positive and negative selection marker. The method begins bycreating an MG1655 ΔthyA strain (LTS00; Table 7) by swapping the genefor an oligonucleotide designed to have 60 bp of homology at thebeginning and the end of the thyA gene. FIG. 5 shows the process bywhich this deletion is performed.

To Delete thyA

This oligonucleotide was ordered as two linear ssDNA fragments fromIntegrated DNA Technologies (Coralville. Iowa). The fragments werehydrated in Qiagen EB buffer (Tris, pH 8, 1.4M NaCl) and mixed at a 1:1ratio. The mixture was then placed in an MJ Research PTC-200 DNA Enginethermocycler that was programmed to heat to 98° C. and then drop thetemperature by 2° C. every 30 seconds until it reached 25° C.

To delete thyA, MG1655+pKD46 (described in Datsenko et al. and Stringeret al., supra) was cultured overnight at 30° C. in LB plus ampicillin(100 μg/ml). The following morning, the overnight culture wassub-cultured 1:100 into 5 ml of fresh LB-ampicillin with 0.2%L-arabinose (w/v) and allowed to grow for approximately three hoursuntil the culture reached an OD₆₀₀ (determined using a HP DU800) of 0.6to 0.8. All proper controls were also taken to validate therecombination event. To prepare the cells for electroporation, the 5 mlinduced culture was split into four 1 ml aliquots and moved to 1.5 mlmicrofuge tubes. The final 1 ml was refrigerated for later analysis orfor further sub-culturing. The microfuge tubes were centrifuged for 60seconds at 14,000 rpm in a cooled (placed in a refrigerator at 2° C.)bench-top microfuge centrifuge (Eppendorf, MiniSpin). The supernatantwas discarded by gently pouring off the liquid and then the pellet wasplaced on ice. The pellet was then re-suspended in 1 ml of chilled ddH2Oand then centrifuged again. This process was repeated once more. Afterthe supernatant is poured off the final time, there is roughly 100 μl ofliquid left in the tube. Next, the cells are re-suspended in theremaining fluid and kept on ice. To this, the prepared linear fragmentis added, in this case the thyA deletion template, variousconcentrations, usually ranging from 200-1000 nmol. This mixture wasthen pipetted into chilled sterile electroporation cuvettes (Bio-Rad,0.1 cm gap). The sample was then electroporated using a Bio-RadMicroPulser set to Ec1 (E. coli, 0.1 cm cuvette, 1.8 kV, one pulse).Next, 1 ml of LB containing ampicillin (50 μg/ml), thymine (100 μg/ml),and trimethoprim (20 μg/ml) (LB-amp-thy-tri) was gently added directlyto the cuvette before incubating the sample at 30° C. with shaking for 3hours. Since the strain now lacks thyA, it is necessary to supplementthe medium with thymine. Trimethoprim acts as a secondary selectorbecause if the strain still contains an active thyA gene, thetrimethoprim is toxic. After that time, the cultures were streaked outonto LB-amp-thy-tri agar plates and allowed to incubate at 30° C.overnight. In addition, 250 μl of each culture were sub-cultured into 5ml of LB-ampicillin-thymine-trimethoprim and incubated overnight at 30°C. with shaking.

In summary, the gene deletion protocol is a two-step process. The firststep uses thyA as a selection marker that disrupts the targeted gene.The second step removes thyA from the genome again, following theprotocol described above. For the first step, strain LTS00 is grownovernight in LB-amp-thy-tri and is sub-cultured 1:100 the followingmorning into 5 ml LB-amp-thy-tri plus 0.2% L-arabinose. These cells areallowed to grow for approximately 6 hours (growth is significantlydiminished when lacking thyA) until the OD₆₀₀ reaches 0.6 to 0.8. Thecells are then prepared for electroporation as described above. Prior toelectroporation, 2 μl of the PCR product containing the thyA gene withhomology to the gene to be deleted is added to the sample.Electroporation follows the protocol described above. Afterelectroporation, 1 ml of LB with ampicillin (50 μg/ml) was added, andthe cells were allowed to incubate for 3 hours at 30° C. with shaking.After that time, the cultures were streaked out onto LB-ampicillin (150μg/ml) agar plates and allowed to incubate at 30° C. overnight. Inaddition, 250 μl of each culture were sub-cultured into 5 ml ofLB-ampicillin (150 μg/ml) and incubated overnight at 30° C. withshaking.

FIG. 6 shows the process by which the selection marker is used to causea gene deletion. Step 1 creates the intermediary thyA+ strain, where thetarget gene has been deleted but the selection marker remains. At thispoint, the cell is able to survive on thymine-depleted medium. Step 2removes the thyA marker so that it can be used again for future genedeletions. The protocol is the same as that for the removal of thyA butthe 120 bp oligonucleotide has homology to the new gene target andremoves thyA and its promoter.

Successful deletion of the gene was confirmed via PCR amplification ofthe deleted region and agarose gel electrophoresis. The amplifiedregions were also sent for genomic sequencing to further confirm thatthe homologous recombination event successfully occurred.

TABLE 6 Deletion Fragments. Deletion Fragments Name Gene Target SequencethyAdt thyA GCAAAATTTCGGGAAGGCGTCTCGAAGAATTTAACGGAGGGTAAAAAAACCGACGCACAC GTGTTGCTGTGGGCTGCGACGATATGCCCAGACCATCATGATCACACCCGCGACAATCAT (SEQ ID NO: 4) metHdt metHTTTGTTGAATTTTTATTAAATCTGGGTTGA GCGTGTCGGGAGCAAGTGCTGGGGTATGACGCGGACTGATTCACAAATCTGTCACTTTTC CTTACAAC (SEQ ID NO: 5) entFdt entFGGCGTACTCTGACACCGACGAATTTTACCC AGTTGCAGGAGGCACACGCGCAACGCTAAACAGGTAAATTAATATTATTTATAAACCCAT AATTAC (SEQ ID NO: 6) tgtdt tgtCGCTGGTTTAAAACGTTGGACTGTTTTTCT GACGTAGTGGAGAAAAACCACCTTTGAACGTTGATTAATATTAATAATGAGGGAAATTTA ATGAGCT (SEQ ID NO: 7) rnrdt rnrGTGGAGTGACGAAAATCTTCATCAGAGATG ACAACGGAGGAACCGAGAAGAAAAAAGTGGCAGAGTGATCAATACCCTCTTTAAAAGAAG AGGGTTA (SEQ ID NO: 8) ycaOdt ycaOTAAAACCCGTATTATTGCGCGCTTTCCGTA CGACTAAAGTGATTTTCGCAGCATTCTGGGCAAAATAAAATCAAATAGCCTACGCAATGT AGGCTTA (SEQ ID NO: 9)

These results demonstrate how the separatome can be defined for achromatographic technique, ion exchange in particular, and can be usedto design and construct novel host cells that have certain genesdeleted, modified, or inhibited. For example, Table 6 describes tenseparate E. coli MG1655 derivatives that have one or more gene deletionsassociated with high affinity host cell proteins. These strains in theircurrent form can be used to express a target recombinant protein andwill have enhanced separation efficiency, column capacity in particular,as these proteins are contained in several fractions of high saltconcentration.

TABLE 7 E. coli Deletion Strains Name Genotype MG1655 Wild Type: F−, λ⁻,rph-1 LTS00 ΔthyA LTS01+ ΔmetH LTS01 ΔthyAΔmetH LTS02+ ΔmetHΔentF LTS02ΔthyAΔmetHΔentF LTS03+ ΔmetHΔentFΔtgt LTS03 ΔthyAΔmetHΔentFΔtgt LTS04+ΔmetHΔentFΔtgtΔrnr LTS04 ΔthyAΔmetHΔentFΔtgtΔrnr LTS05+ΔmetHΔentFΔtgtΔrnrΔycaO

Table 8 lists high priority genes for DEAE ion exchange media. Thistable was generated by analyzing the DEAE separatome database with theimportance score (Equation 3). In this iteration of analyisis, the ISvariables were specifically defined as follows. The summation includedNaCl fractions 60 mM, 109 mM, 159 mM, 208 mM, 258 mM, 307 mM, 357 mM,406 mM, 456 mM, 505 mM, 1000 mM, b1=1, y_(max)=1000 mM; for h_(i,j),h_(i,total), and h_(j,total), a count of distinct peptides identified inthe sample was used to indicate amount of protein; MWref=170 kDa (themolecular weight of the largest gene product, mukB); α=1.

Future strains of the LTS series of Table 7 will have additional genes,alone or in various combinations, identified in Tables 8 and 9, deleted,modified, and/or inhibited as the recovery capacity is pushed towardhigher values.

TABLE 8 High Priority Genes of the DEAE Separatome, Loading pH 7 GeneName rpoC rpoB hldD metH entF mukB tgt rnr glgP recC ycaO glnA ptsI metEsucA hrpA groL gatZ speA thiI nusA tufA degP clpB rapA metL ycfD nagDilvA fusA cyaA gldA dnaK ygiC gyrA glnE carB ppsA degQ usg ilvB thrSrecB entB dusA typA prs cysN atpD purL

The high priority genes in Table 8 are listed in descending rank order,from greater importance to lesser importance, according to theirimportance score as calculated using Equation 3.

TABLE 9 Further High Priority Genes of the DEAE Separatome, Loading pH 7Gene Name hldD cutA rraA usg tufA nagD ycfD ptsI gldA slyD speA prs tgtargG glnA rpoB hemL groL rpoC metE typA entB fusA csrA gatZ

The high priority genes in Table 9 are listed in descending rank order,from greater importance to lesser importance, according to theirimportance score as calculated using Equation 3. The summation includedNaCl fractions 60 mM, 109 mM, 159 mM, 208 mM, 258 mM, 307 mM, 357 mM,406 mM, 456 mM, 505 mM, 1000 mM, b1=1, y_(max)=1000 mM. For h_(i,j),h_(i,total), and h_(j,total), mass spectroscopy data were used todetermine the amount of protein, which was defined as the number ofconfident sequencing events that matched to peptides associated with agiven protein (h_(i)). MWref=170 kDa (the molecular weight of thelargest gene product, mukB); α=0. Six genes listed in Table 9 are uniquecompared to those listed in Table 8: cutA, rraA, slyD, argG, hemL, andcsrA.

The genes listed in Tables 8 and 9 have been determined by the inventorsto represent preferred/suitable genes to target for deletion, etc., forimproving the chromatographic separation efficiency of target host cellor target recombinant peptides, polypeptides, or proteins expressed inthe E. coli host cells disclosed herein via DEAE anion exchangechromatography adsorbed at pH 7 and 60 mM NaCl.

As would be apparent to one of ordinary skill in the art, the geneslisted in each of Tables 8 and 9 can advantageously be used alone, ortogether with one another in various combinations, to improvechromatographic separation efficiency of peptides, polypeptides, andproteins expressed in E. coli host cells. In addition, the genes listedin each of these two tables can further advantageously be used invarious combinations with one another as well.

The number of such combinations of genes, either for Table 8 alone,Table 9 alone, or Tables 8 and 9 together, is determined by thecombination equation:

$\begin{matrix}\frac{n!}{{r!}{\left( {n - r} \right)!}} & {{Equation}\mspace{14mu} 6}\end{matrix}$

-   -   where n is the set of genes out of which selection occurs (the        unique set of genes from Table 8 or Table 9 taken alone or in        combination, i.e., without repetition), and    -   r is the number of genes selected for deletion, modification,        and/or inhibition together.

The number of combinations of n genes selected r at a time is equal to nfactorial divided by r factorial multiplied by n minus r factorial, or

nCr=n!/(r!((n−r)!))  Equation 7

-   -   where nCr represents the number of possible unique combinations        of gene selections from a master group with n distinct genes.

The number of genes (r) selected can be 2 or more, 3 or more, 4 or more,5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 ormore, 12 or more, and so on, encompassing all the genes listed in Tables8 and 9. By way of example only and not limitation, the number of genes(r) selected can be in ranges from 1-12 and any range therein, includingthe end points; 2-12 and any range therein, including the end points;3-12 and any range therein, including the end points; 4-12 and any rangetherein, including the end points; 5-12 and any range therein, includingthe end points: 6-12 and any range therein, including the end points;7-12 and any range therein, including the end points; 8-12 and any rangetherein, including the end points; 9-12 and any range therein, includingthe end points; 10-12 and any range therein, including the end points;or 11-12. Twelve is merely an illustrative upper limit: upper limits foreach table include all the listed genes, including any range of genestherein.

As a non-limiting example, selecting 5 genes for deletion from thoselisted in Table 8 and using the combination equation, i.e., when nequals 50 and r equals 5, results in 2,118,760 unique combinations, oneof which includes five genes in the list of non-essential genespublished by Gerdes et al. (2003), discussed below in Section II.

As the genes listed in Tables 8 and 9 have been identified as highpriority candidates for deletion, modification, or inhibition toconstruct improved E. coli host cell strains for target peptide,polypeptide, and protein expression and purification, it is highlylikely, and fully expected, that most, if not all, combinations of thesegenes will be effective in improving separation efficiency of targetbiomolecules from host cells in which these biomolecules are expressedand in which combinations of these genes are deleted, modified, orinhibited.

Deletion, modification, and/or reduction/total inhibition of expressionof various combinations of genes listed in Tables 8 and 9 as calculatedabove includes both sequential (contiguous) and non-sequential(non-contiguous) combinations (which involve “skipping” or omittinglisted genes), as well as random combinations of high ranking geneslisted in these Tables, i.e., any combinations predicted by equations 6and 7. Essential genes that can be modified by the methods discussedbelow can also be included in any of these gene combinations whennecessary.

A consideration in designing such combinations involves geneessentiality. Essential genes can be deleted, etc., if the modified hostcells exhibit acceptable viability, growth rates, protein expressionlevels, etc., for the intended application. Alternatively, essentialgenes can be modified, for example, by reducing their expression byreplacing their naturally occurring promoters with weaker promoters,introducing strategic point mutations to replace amino acids involved inresin binding while still maintaining satisfactory levels ofgene/protein activity, or replacing endogenous E. coli genes with genesfrom other organisms that perform the same or similar functions and thatdo not significantly adversely affect chromatographic separationefficiency and separation capacity, or cell growth, viability, andcapacity for expression, rather than deleting them entirely. Suchreplacement genes include heterologs, homologs, analogs, paralogs,orthologs, and xenologs. These strategies facilitate improvements inchromatographic separation efficiency even when interfering host cellproteins are expressed from essential genes. In addition, as discussedabove in the definition of “essential genes”, various feeding strategiescan be used in the present host cells and methods to circumventpotentially deleterious effects due to deletion, etc., of essentialgenes that would otherwise adversely impact chromatographic separationefficiency if present.

The effectiveness of any of the various possible combinations of genestargeted for deletion, selected from either Table 8 alone, Table 9alone, or Tables 8 and 9 together, as described above in improvingchromatographic separation efficiency of target host cell or targetrecombinant peptides, polypeptides, and proteins can be determinedwithout undue experimentation by the methods disclosed herein.

Section II.

Cloning Strains and Vectors: E. coli Strain K-12

Construction of Knockout Strain K-12 MG1655

Parent Cell Line Selection

There are two predominant strain derivatives within E. coli: theB-strains and the K-12-strains, with the ones used herein to demonstrateseparatome principles being B-strain BL21 (DE3) (note Section I, above)and K-12-strain MG1655, exemplified in this section.

While the data generated in Section I. described above were based on theproteome of BL21, it was desirable to build a knockout strain in theK-12 derivative MG1655 as well. However, it should be noted that besidesK-12 strain MG1655, many other strains can be used in embodiments ofthis invention, including those listed in, and immediately below, Table1.

Fortunately, the differences between the two strains are minimal. Themost apparent difference between the two strains is that the BL21(DE3)strain has the T7 RNA polymerase incorporated into its genome under thecontrol of the lac repressor, allowing for the use of T7 promoters fortight control of recombinant expression (Studier and Moffatt (1986),Journal of Molecular Biology 189 (1): 113-30). Other than thatmodification, the two strains are otherwise highly similar. In fact, theB and K genomes align with greater than 99% base-pair matching overapproximately 92% of their genome (Jeong (2009), Journal of MolecularBiology 394(4): 644-652). The remaining un-matched segments can bemostly accounted for as insertion sequences, and the remainingdifferences are a few full-gene deletions and single-nucleotidepolymorphisms that cause frame shifts (Studier, (2009) Journal ofMolecular Biology 394(4): 653-80). While these differences areinteresting in the scope of phylogenetics, they are minimal enough thatthey have little to no impact on the proteomics of the cell lines.

Genes Selected for Deletion in the K-12 Strain

Five non-essential genes (from among a list of non-essential genespublished by Gerdes et al. (2003) J. Bacteriol. 185, (19): 5673-5684)were selected for deletion from the K-12 genome. These were metH, entF,tgt, rnr, and ycaO. Using the importance score calculated from 59-1000mM according to importance score Equation 3, the deleted genes rank:tgt: 13, entF: 40, ycaO: 34, metH: 67, and rnr: 120. Lower rank numbersare more important than higher rank numbers, i.e., lower rank numbersreflect higher importance scores. The primers used for gene deletion andthe double stranded DNA deletion templates are shown in Table 10.

TABLE 10 Deletion Templates and Primers Base Name Pairs SequenceDeletion Templates metHdt3 98GTT GTA AGG AAA AGT GAC AGA TTT GTG AAT CAG TCC GCGTCA TAC CCC AGC ACT TGC TCC CGA CAC GCT CAA CCC AGA TTT AAT AAA AAT TCA ACA AA (SEQ ID NO: 10) metHdt5 98TTT GTT GAA TTT TTA TTA AAT CTG GGT TGA GCG TGT CGGGAG CAA GTG CTG GGG TAT GAC GCG GAC TGA TTC ACA AATCTG TCA CTT TTC CTT ACA AC (SEQ ID NO: 11) entFdt3 96GTA ATT ATG GGT TTA TAA ATA ATA TTA ATT TAC CTG TTTAGC GTT GCG CGT GTG CCT CCT GCA ACT GGG TAA AAT TCGTCG GTG TCA GAG TAC GCC (SEQ ID NO: 12) entFdt5 96GGC GTA CTC TGA CAC CGA CGA ATT TTA CCC AGT TGC AGGAGG CAC ACG CGC AAC GCT AAA CAG GTA AAT TAA TAT TATTTA TAA ACC CAT AAT TAC (SEQ ID NO: 13) tgtdt3 97AGC TCA TTA AAT TTC CCT CAT TAT TAA TAT TAA TCA ACGTTC AAA GGT GGT TTT TCT CCA CTA CGT CAG AAA AAC AGTCCA ACG TTT TAA ACC AGC G (SEQ ID NO: 14) tgtdt5 97CGC TGG TTT AAA ACG TTG GAC TGT TTT TCT GAC GTA GTGGAG AAA AAC CAC CTT TGA ACG TTG ATT AAT ATT AAT AATGAG GGA AAT TTA ATG AGC T (SEQ ID NO: 15) rnrdt3 97TAA CCC TCT TCT TTT AAA GAG GGT ATT GAT CAC TCT GCCACT TTT TTC TTC TCG GTT CCT CCG TTG TCA TCT CTG ATGAAG ATT TTC GTC ACT CCA C (SEQ ID NO: 16) rnrdt5 97GTG GAG TGA CGA AAA TCT TCA TCA GAG ATG ACA ACG GAGGAA CCG AGA AGA AAA AAG TGG CAG AGT GAT CAA TAC CCTCTT TAA AAG AAG AGG GTT A (SEQ ID NO: 17) ycaOdt3 97TAA GCC TAC ATT GCG TAG GCT ATT TGA TTT TAT TTT GCCCAG AAT GCT GCG AAA ATC ACT TTA GTC GTA CGG AAA GCGCGC AAT AAT ACG GGT TTT A (SEQ ID NO: 18) ycaOdt5 97TAA AAC CCG TAT TAT TGC GCG CTT TCC GTA CGA CTA AAGTGA TTT TCG CAG CAT TCT GGG CAA AAT AAA ATC AAA TAGCCT ACG CAA TGT AGG CTT A (SEQ ID NO: 19) Primers metH-F 68TTT GTT GAA TTT TTA TTA AAT CTG GGT TGA GCG TGT CGGGAG CAA GTG CGC CAC CCA TCA CAG CTT TA (SEQ ID NO: 20) metH-R 70GTT GTA AGG AAA AGT GAC AGA TTT GTG AAT CAG TCC GCGTCA TAC CCC AGG GGA AGG CGT CTC GAA GAA T (SEQ ID NO: 21) entF-F 66GGC GTA CTC TGA CAC CGA CGA ATT TTA CCC AGT TGC AGGAGG CAC ACG CCA CCC ATC ACA GCT TTA (SEQ ID NO: 22) entF-R 70GTA ATT ATG GGT TTA TAA ATA ATA TTA ATT TAC CTG TTTAGC GTT GCG CGG GGA AGG CGT CTC GAA GAA T (SEQ ID NO: 23) tgt-F 67CGC TGG TTT AAA ACG TTG GAC TGT TTT TCT GAC GTA GTGGAG AAA AAC GCC ACC CAT CAC AGC TTT A (SEQ ID NO: 24) tgt-R 70AGC TCA TTA AAT TTC CCT CAT TAT TAA TAT TAA TCA ACTTTC AAA GGT GGG GGA AGG CGT CTC GAA GAA T (SEQ ID NO: 25) rnr-F 67GTG GAG TGA CGA AAA TCT TCA TCA GAG ATG ACA ACG GAGGAA CCG AGC GCC ACC CAT CAC AGC TTT A (SEQ ID NO: 26) rnr-R 70TAA CCC TCT TCT TTT AAA GAG GGT ATT GAT CAC TCT GCCACT TTT TTC TTG GGA AGG CGT CTC GAA GAA T (SEQ ID NO: 27) ycaO-F 67TAA AAC CCG TAT TAT TGC GCG CTT TCC GTA CGA CTA AAGTGA TTT TCC GCC ACC CAT CAC AGC TTT A (SEQ ID NO: 28) ycaO-R 70TAA GCC TAC ATT GCG TAG GCT ATT TGA TTT TAT TTT GCCCAG AAT GCT GCG GGA AGG CGT CTC GAA GAA T (SEQ ID NO: 29)

The FRUIT method (Flexible Recombineering Using Integration of thyA) asdescribed by Stringer et al. ((2012) PloS one. 7(9):e44841) was selectedas the gene deletion method for this example for several reasons.

The predominant homologous recombination method utilizes antibioticresistance as the selectable deletion marker, but maintenance of thepKD46 plasmid requires the presence of ampicillin. This requirement thennecessitates using a different antibiotic resistance as the selectionmarker and growing newly transformed cells in the presence of twodifferent antibiotics during selection (in the case that it is desirablefor the plasmid to be maintained, as in this instance). Growth in thepresence of dual antibiotic selection is very hard on the cells postelectroporation, and additionally can cause severe growth inhibition.The growth inhibition causes the cloning process to be very slow, inaddition to making it difficult to select for the clones that still havefavorable growth characteristics.

Secondly, antibiotic selection markers are positive selectors only,meaning that one can only select for the presence of the resistancegene, not for the absence. After the selection marker is removed, clonesmust be selected by growth on antibiotic-deficient agar overnight, andthen replica plated onto agar containing antibiotics and incubated againovernight. After the second overnight growth, positive clones can beidentified on the first plate by the absence of growth on the second.This process takes two days for clone selection, and there is room forsignificant error when replica plating.

The FRUIT method utilizes gene thyA as the selection marker, which canbe positively and negatively identified by the lack of thymine or theinclusion of trimethoprim, respectively. The thyA gene is returned tothe genome when gene deletion is finalized, so that the final knockouthost strains contain this gene. Additionally, this method requires asingle plating step for clone selection for both the inclusion andremoval of the marker.

The gene deletions were confirmed by PCR as well as by DNA sequencing.

FIG. 7 shows the electrophoresis of the PCR amplification of each targetgene after deletion. As shown, genes metH, entF, tgt, rnr, and ycaO allhave 120 bp bands, which correspond to the inserted deletion fragment.The lane corresponding to tgt also shows a larger band that correspondswith the intermediate deletion step where thyA is inserted as part ofthe knockout process. This indicates that there is a mixture of the twoclones present, and further agar plating on trimethoprim and selectionneeds to be performed to select for the completed clone. The smaller tgtband, in addition to the other single bands, was isolated and sent forDNA sequencing. DNA sequencing confirmed the presence of the deletiontemplate, which confirms that the targeted genes were successfullyremoved from the chromosome.

Growth Studies

Fed-batch growth studies of K-12 knockout strain LTS05+t were conductedto determine whether growth had not been considerably diminished (i.e.,whether doubling time had increased by greater than 5%) in comparison tothe parent strain MG1655. Both the LTS05+t strain and the MG1655 straincontained pKD46 (amp^(r)) plasmid and were grown in the presence ofampicillin to ensure selection of the desired strain, but neither strainwas induced during growth. FIG. 8 shows the results of these two growthstudies, where Bug Units, as defined by the BugEye Biomass monitor, arearbitrary optical density units that allow the user to monitor therelative growth of cultures. These units are dependent on growthconditions and linearly correlate to optical density units as measuredby a spectrophotometer (OD₆₀₀)

As shown in FIG. 8, the lag phase of LTS05+t was roughly half that ofMG1655. Once the growth entered log phase, both cultures grew at roughlythe same rate as evaluated by comparing the slope of the curves. Fromtime point 6:00 to 9:00, the slope of growth for LTS05+t was 0.212,whereas the slope of growth for MG1655 was 0.225 from time point 8:00 to11:00. While exponential growth was maintained in the wild-type forroughly 10 hours and reached a final OD₆₀₀ of 48.8, LTS05+t grewexponentially for approximately 15 hours, reaching a final OD₆₀₀ of 92,almost double that of the parent strain. This growth difference couldresult from the tgt knockout, as previously mentioned by Noguchi et al.(1982) J. Biol. Chem. (11): 6544-6550.

The shortened lag phase of LTS05+t was confirmed by two separate growthstudies, the first run being the fed-batch discussed above, and thesecond run being a standard batch.

These data are shown in FIG. 9, where it is evident that the transitionfrom lag phase to log phase occurs at the same time for both batch andfed-batch fermentation.

These data demonstrate that the gene deletions do not cause asignificant reduction in cellular growth and function. In fact, growthof deletion strain LTS05+t was actually improved under fed-batchconditions.

Column Capacity Measurements

The mass spectroscopy data disclosed above in Section I. provide proteinquantitation through the spectral counting method. These data can beutilized to provide a rough estimation of column capacity improvement(Total Contaminant Pool: TCP) as well as the reduction of protein boundin each individual Elution Contaminant Pool (ECP) by examining thechanges in theoretical protein concentration as targeted genes areremoved. The results of these calculations are shown in Table 11.

TABLE 11 Predicted Improvement for the Five Selected Gene Knockouts TCPand ECP Improvement Total Contaminant 2.7% Pool  60 mM 0.7% 109 mM 1.2%159 mM 2.1% 208 mM 3.8% 258 mM 1.0% 307 mM 2.9% 357 mM 4.6% 406 mM 7.2%456 mM 4.5% 505 mM 3.9% 1000 mM  1.2%

Total Contaminant Pool Assessment

Modifications to the total contaminant pool (TCP) were measured bydetermining the percent of proteins that bound to DEAE under variousloading conditions. This was accomplished by applying 40 mg of totalprotein to the column under binding conditions while collecting the flowthrough. The binding conditions used were 25 mM Tris, pH 7, at a saltconcentration of 159 mM NaCl, which represents a typical columnoperating condition as used by commercial manufacturers in theirpurification processes. The bound proteins were then eluted using 1MNaCl, and the peak was collected. Two runs were completed at each saltconcentration, per cell line, to verify the data.

A BioRad DC assay was used to determine the total protein in eachcollected fraction and thus determine the percent protein bound to theresin. The BioRad DC assay is a colormetric assay, requiring a standardcurve to be utilized to determine protein concentration. A new BSAstandard curve was built using the same Fast Protein LiquidChromatography (FPLC) buffers as the solvent. The readable range for theassay is between 0.1 and 1 for the A₇₅₀, but is most accurate between0.1 and 0.5 (0.2 to 2 mg protein). Since most of the samples wereconcentrated, they required dilution for the assay. To improve accuracyof the assay, samples were assayed at four different dilutions (oftenbetween 1× and 20×), and the measurements that fell within the readablerange were used to calculate the protein concentration, which was thenaveraged with themselves. The FPLC runs were repeated twice. Where thedata were averaged, they are presented in and Table 12 as a %/avg. Ifonly one point was used, it was because the other point was well outsidethe readable range of (0.1-0.5).

TABLE 12 Column Capacity at 159 mM Salt Data Average Strain Points Bound% Error LTS05 + t 3 20.6% ±1.1% MG1655 1 30.7% Measured Improvement10.1% ±1.1 Predicted Improvement 1.6%

Under these conditions, 30.7% of the proteins from the control (MG1655)bound to the resin. In contrast, 20.6% of the proteins from deletionstrain LTS05+t bound the resin, representing a significant improvement(approximately 10%) in Total Column Capacity.

When compared to the theoretical TCP improvement of 2.7% (as shown inTable 10), it can be seen that the improvement is in the same order ofmagnitude. The gel images of the bound proteins for these twoexperiments show little difference (data not shown). This is likelybecause there are multiple proteins at the same molecular weight as thedeleted proteins in the knockout strain.

These results demonstrate that the data from Section I. can be used topredict downstream column capacity improvements with relative accuracy,i.e., within the same order of magnitude.

Eluting Contaminant Pool Assessment

In addition to the measurement of column capacity changes due to TotalContaminant Pool reduction, it is important to recognize that genedeletions also change the Eluting Contaminant Pool (ECP). The assessmentof the ECPs was measured using the same protocol and salt windows asdescribed in Section I. Again, the column was loaded at 10% breakthroughto simulate commercial practices. The resulting samples wereconcentrated using a Vivaspin2 (GE Healthcare, 28-9322-40) with a 5 kDacutoff to a final volume of approximately 200 μl. The samples were thenanalyzed by BioRad DC assay to determine total protein content, andloaded on an SDS-PAGE gel for further analysis.

The results of the ECP measurements for LTS05+t are shown in FIG. 11,and the results of the ECP measurements for wild-type MG1655 are shownin FIG. 12. The top portion of each figure shows the Fast Protein LiquidChromatography chromatogram, with the A₂₈₀ on the left axis and % bufferB on the right axis. Below each chromatogram is a table showing the %buffer B converted into a mM salt concentration, followed by themeasured protein concentration in the window. As each of these stepfractions was 15 ml of total volume, it was necessary to concentrate thesamples prior to further analysis. The samples were concentrated using a5 kDa VivaSpin2 concentrator to reduce the final volume to approximately200 μl. It is important to note that due to the high absorbance readingof the A₂₈₀ (outside of the linear correlation described by Beer's law)and the presence of DNA and RNA in the sample, it cannot be used as anaccurate measure of protein concentration. Instead, a BioRad DC assaywas used to determine the protein content of each sample, but theminimum threshold for this assay is around 0.2 mg/ml. Even afterconcentration, a few of the samples had a protein concentration belowthe readable range for the assay and are noted at 0 mg/ml despite theA₂₈₀ and subsequent SDS-PAGE indicating otherwise. The next row shows abreakdown of how all of the applied proteins distributed over theelution windows as a percentage calculated as mg protein in elutionwindow/total mg of protein applied to the column. Note that while thepercent of protein unbound seems higher than what was measured in theTCP binding experiments, this is due to the 10% overloading of thecolumn. The fourth row focuses on the breakdown of just the boundprotein into specific windows, and gives a percentage calculated as mgprotein in the window/mg protein in all bound fractions (59.5 mM to 1000mM). This number provides the best indication of how the ECP changed inknockout strain LTS05+t.

Finally, the corresponding lane from the SDS-PAGE gel is shown at thebottom. This is particularly important for the samples that were belowthe readable range for the protein concentration assay. Each lane of theprotein gel was loaded with 25 μg of proteins, for the samples where theprotein assay worked, or at the maximum volume possible for the samplesthat were too dilute to assay. It is important to keep in mind that eachband in the protein gel may consist of multiple proteins due to thelarge variety of proteins being eluted in each window (from as many as300 to as few as 70).

If the percent of bound protein for both knockout strain LTS05+t andparent MG1655 are added cumulatively (FIG. 13), the result is a measureof the column loading profile. As shown in FIG. 13, the knockout strainLTS05+t has a reduced column binding in the earlier windows, whichmatches the predicted ECP reductions due to the gene deletions.

The knockout strain shows decreased percentage of proteins eluting inthe 60 mM and 109 mM windows, and it also appears to have a reducednumber of proteins in the 307 mM window. Additionally, by comparing thegels, the number of bands in the 456 mM window decreases from four toone (FIGS. 11 and 12). Further analysis of the elution windows byLC-MS/MS would be able to indicate where the energy was shiftedmetabolically and what protein concentrations were increased ordecreased based upon the original deletions.

The results presented herein demonstrate that selective gene deletionsin host cells that can be used to produce target peptides, polypeptides,and proteins of interest not only result in an improvement in overallcolumn capacity, but additionally simplify specific elution windows.Exploitation of these phenomena by the concepts and methods exemplifiedherein will significantly improve overall purification, i.e., totalrecovery as well as level of purity, of target peptides, polypeptides,and proteins of interest from host cells more quickly and moreeconomically than is possible using conventional host cells andchromatographic purification methods.

Example 3 Data-Informed Construction of Escherichia coli for ImprovedBioseparation: Construction of an E. coli Cell Line Having a Reduced Setof Host Cell Proteins Associated with DEAE Ion Exchange Chromatography

A key step and potential bottleneck associated with the expression andisolation of a recombinant product is the initial chromatography captureof the protein, for as the mixture containing the target or desiredprotein passes over a chromatography resin, host cell proteins (HCPs)and the target compete for binding sites. This competition reducescolumn efficiency, and due to the binding/elution of HCPs, requiresadditional purification steps. This is especially problematic in theproduction of a biotherapeutic, for example, because the exactingrequirements of purity and efficacy can require multiple purificationsteps, and as these steps become numerous, reduce the overall efficiencyof the process. While work can be done to tailor a downstream processingregimen for a particular recombinant product, modern techniques likebioinformatics, computational genomics, and proteomics can be harnessedto improve the basic knowledge and design of the cell line used inrecombinant manufacturing. Anticipated benefits of exploiting thesetechniques would be the development of a series of cell lines optimizedfor a method of purification that does not require the use of anaffinity tail or biospecific interaction to achieve a high degree ofefficiency.

This example describes the mapping of the E. coli chromosome to discernrelationships between the loci of genes of key nuisance proteins (e.g.,those in large concentration and/or of high binding affinity) associatedwith chromatographic techniques, for example DEAE ion exchange andImmobilized Metal Affinity Chromatography, to guide host cell linedevelopment with a reduced number of HCPs that are widely applicable torecombinant peptide, polypeptide, and protein production irrespective ofthe exact target molecule, exemplifying the separatome concept. This isan example of improving separation capacity for a chromatographicresin/buffer combination in the absence of expression of a recombinantpeptide, polypeptide, or protein.

Materials and Methods

Host Cells

Wild type E. coli MG1655 (parent cell) was obtained from the Yale ColiGenetic Stock Center (New Haven, Conn.). The E. coli host cell linederived from this parent cell, having a reduced set of HCPs associatedwith DEAE ion exchange chromatography via selective gene deletions ofrfaD, usg, rraA, cutA, nagD, and speA, is designated LTSF06. In thisexample, LTSF06 does not express any recombinant peptide, polypeptide,or protein.

Primers

The primers used for gene deletions are based on those developed anddescribed in the Keio collection (Baba et al. (2006) Mol Systems Biol.2: 2006.0008, doi:10.1038/msb4100050). Further information concerningthe Keio collection is stored in their online data repository, GenobaseVer. 8, located online at http://ecoli.naist.jp/GB8-dev/index.jsp.

TABLE 13 Primers used for gene deletion and PCR confirmation Primer nameSequence rfaD-F_Frt GCAAAACCAACATCCGCCATGAAGGACTAGCTAAAACCCAAACTAGTTTGGTGTAGGCTGGAGCTGCTTC (SEQ ID NO: 30) rfaD-R_FrtCCGGTGCCATCACATCGATTATCGCCTGGGGATAGCGC GCCTGGAGCGTGATGGGAATTAGCCATGGTCC(SEQ ID NO: 31) rfaD-F2 GCAAAACCAACATCCGCCAT (SEQ ID NO: 32) rfaD-R2CCGGTGCCATCACATCGATTA (SEQ ID NO: 33) usg-F_FrtCGGCATCATTGCTGTGTAAACTGGGTTTTAACGCCGTT CATCATCCGGCAGTGTAGGCTGGAGCTGCTTC(SEQ ID NO: 34) usg-R_Frt GAAGACGGTGATGGGTTCGTTCGCCACCTGGGAGAGCGCCTTTTCCAGCTATGGGAATTAGCCATGGTCC (SEQ ID NO: 35) usg-R2GCGGCATCATTGCTGTGTAA (SEQ ID NO: 36) usg-F2 GAAGACGGTGATGGGTTCGT(SEQ ID NO: 37) rraA-F_Frt CGTACTGTCAAGGGAGCGTTACTGACTAACCTGCTGTTTGTTTTAGGGATGTGTAGGCTGGAGCTGCTTC (SEQ ID NO: 38) rraa-R_FrtGAGCTGGAGCGTGAGAACAACCATCTGAAAGAACAGCA GAACGGCTGGCAATGGGAATTAGCCATGGTCC(SEQ ID NO: 39) rraA-F2 CGTACTGTCAAGGGAGCGTT (SEQ ID NO: 40) rraA-R2AAGAGCTGGAGCGTGAGAAC (SEQ ID NO: 41) cutA-F_FrtCGACTAACATCCTTCCCCCGTCCGTTGTATAGTGACC TCTCTCTTGCGGTGTGTAGGCTGGAGCTGCTTC(SEQ ID NO: 42) cutA-R_Frt AAAGCAAAGGCTTGATCCGCGGGGACAAATTGTGAACGTCCCGGCGCGTCATGGGAATTAGCCATGGTCC (SEQ ID NO: 43) cutA-F2CGACTAACATCCTTCCCCCG (SEQ ID NO: 44) cutA-R2 AAAGCAAAGGCTTGATCCGC(SEQ ID NO: 45) nagD-F_Frt TTGGAGCGTCAGCATTCACTGCTGGAAAATCCATGTGCTTATGGGTTGTTGTGTAGGCTGGAGCTGCTTC (SEQ ID NO: 46) nagD-R_FrtTATTGCAGGAGCTGCGTAGGCCTGATAAGCGTAGCGC ATCAGGCAGTTTGATGGGAATTAGCCATGGTCC(SEQ ID NO: 47) nagD-F2 CGTTTCGCACTAATCTGCCG (SEQ ID NO: 48) nagD-F2CGTTTCGCACTAATCTGCCG (SEQ ID NO: 49) speA-F_FrtTTGGAGCGTCAGCATTCACTGCTGGAAAATCCATGTG CTTATGGGTTGTTGTGTAGGCTGGAGCTGCTTC(SEQ ID NO: 50) speA-R-Frt CGACGAGGAAGGGTTGGATTTGTCACAATAAATTGTGGCGGATTATCACCATGGGAATTAGCCATGGTCC (SEQ ID NO: 51) speA-F2TTGGAGCGTCAGCATTCACT (SEQ ID NO: 52) speA-R2 CGACGAGGAAGGGTTGGATT(SEQ ID NO: 53)

“Gene”-F-Frt are the forward primers with homology to antibioticresistant cassettes with FRT sites specific to the gene of interest.“Gene”-R-Frt are the reverse primers with homology to antibioticresistant cassettes with FRT sites specific to the gene of interest. Thefinal 20 bp portion of the sequence is the part homologous to pKD3 orpKD4, which are plasmids containing the antibiotic resistance cassette.“Gene”-F2 are the forward primers with homology to the gene of interestand homology to the gene specific FRT-antibiotic resistant cassette,depending on the design. “Gene”-R2 are the reverse primers with homologyto the gene of interest and homology to the gene specific FRT-antibioticresistant cassette, depending on the design.

Construction of the Knockout Strain

The DEAE separatome database presented in Example 2 was utilized toprovide the raw protein data needed for the importance scorecalculation. Six non-essential genes were selected for deletion from thegenome based on their importance score (IS) determined from importanceequation 3:

$\begin{matrix}{{importance}_{i} = {\sum_{j}\; \left\lbrack {{b_{1}\left( \frac{y_{cj}}{y_{\max}} \right)}\left( \frac{h_{i,j}}{h_{i,{total}}} \right)\left( \frac{h_{i,j}}{h_{j,{total}}} \right)\left( \frac{{MW}_{i}}{{MW}_{ref}} \right)^{\alpha}} \right\rbrack_{i}}} & \;\end{matrix}$

The summation included NaCl fractions 60 mM, 109 mM, 159 mM, 208 mM, 258mM, 307 mM, 357 mM, 406 mM, 456 mM, 505 mM, 1000 mM, b1=1, y_(max)=1000mM.

For h_(i,j), h_(i,total), and h_(j,total), the mass spectroscopy datathat reported the number of confident sequencing events that matched topeptides associated with the given protein was used to indicate amountof protein; MWref=170 kDa (the molecular weight of the largest geneproduct, mukB); α=1.

The genes selected for deletion in this example were rfaD, usg, rraA,cutA, nagD, and speA.

Knockouts were performed via homologous recombination according to theprotocol described by Datsenko and Wanner (Datsenko et al. (2000) PNAS97(12):6640-5), which utilizes the Lambda Red system in conjunction withFLP-FRT recombination to remove the desired genomic regions.Confirmation of gene deletions was determined by PCR.

Fed-Batch Cultivation

To start a fermentation, a 3 L Applikon® bioreactor (Foster City,Calif.) was charged with 1 L of M9 salts, 1 ml silicone anti-foam, 10g/l glucose, and ampicillin (150 μg/ml). The reactor was inoculated with100 ml of culture grown for eight hours in M9 medium. Prior toinoculation, culture broth was centrifuged and resuspended in freshmedium. During growth, adjustments in oxygen delivery and agitation ratewere made as necessary to ensure that the dissolved oxygen concentrationdid not drop below 35%. The pH was maintained at approximately 6.8during the cultivation by adding 7M NH₄OH as needed, and the temperaturewas maintained at 37° C. using a heating jacket and cooling loop. Anexponential feeding profile of glucose (500 g/l) was based on that of acollaborator (Mckinzie Fruchtl), originally proposed by Korz et al.(1995) Journal of Biotechnology 39(1):59-65 and Lee et al. (1996) Trendsin Biotechnology 14(3):98-105.

At the end of the fermentation process, cells were harvested viacentrifugation at 12,000×g for 30 minutes at 5° C. (Beckman CoulterAvanti, JLA-10.500 fixed angle rotor). Optical densities were monitoredusing a Bugeye optical density probe (BugLab, Foster City, Calif.),providing a measurement of arbitrary gowth units, and a DU800 BeckmanCoulter spectrophotometer (Brea, Calif.), providing growth measurementsas OD₆₀₀.

Lysate Preparation

A 50 g cell pellet was re-suspended in 150 ml of 25 mM Tris buffer, pH7, 1 mM phenylmethylsulphonyl fluoride (PMSF), 20 μg/ml aprotinin, and 1mM ethylenediamine-tetraacetic acid (EDTA), sonicated, and clarified bycentrifugation at 50,000×g for 30 minutes followed by filtration througha 0.45 μm SUPOR® membrane to produce the lysate applied to the column.

Column Capacity Measurements

Chromatography was performed using an ÄKTA FPLC. Diethylaminoethylcellulose (DEAE) was selected as the ion exchange (IEX) resin due to itsprevalence of use in manufacturing; specifically, the column used was a1 ml HiTrap DEAE FF from GE (Piscataway, N.J.). 25 mM Tris buffer, pH 7,was selected for all of the FPLC purification steps. The loading buffercontained 10 mM NaCl to minimize non-specific binding (Buffer A). Theelution buffer contained 2M NaCl, which is sufficient to desorb boundproteins (Buffer B). The flowrate for all FPLC experiments was set to 1ml/min. Prior to loading the column with lysate, the system was washedwith 10 column volumes (CVs) Buffer A. The column was loaded at 10%breakthrough, and then washed with 10 column volumes (CV) of Buffer A toremove any unbound proteins.

Reductions in the amount of HCPs that natively bind were measured bydetermining the percent of proteins that bound to DEAE under variousloading conditions. This was accomplished by applying 40 mg of totalprotein to the column under binding conditions while collecting the flowthrough. The binding conditions used were 25 mM Tris, pH 7, at saltconcentrations of 5 mM, 100 mM, and 250 mM NaCl. The bound proteins werethen eluted using 2M NaCl and the peak was collected. Three runs werecompleted at each salt concentration to verify data. A BioRad DC™Protein assay was used to determine the total protein in each collectedfraction and thus determine the percent protein bound and unbound.

Results and Discussion

To construct an E. coli cell line that has a reduced set of HCPsassociated with DEAE ion exchange chromatography, we analyzed the DEAEseparatome database, developed in Example 2, using the Importance Score(IS) equation (Equation 3).

$\begin{matrix}{{importance}_{i} = {\sum_{j}\; \left\lbrack {{b_{1}\left( \frac{y_{cj}}{y_{\max}} \right)}\left( \frac{h_{i,j}}{h_{i,{total}}} \right)\left( \frac{h_{i,j}}{h_{j,{total}}} \right)\left( \frac{{MW}_{i}}{{MW}_{ref}} \right)^{\alpha}} \right\rbrack_{i}}} & {{Equation}\mspace{14mu} 3}\end{matrix}$

The IS provides the ability to quickly interpret chromatography data andrank HCPs to indicate their effect on column capacity. Candidate genesfor knockout, etc., can be selected based on the IS and data onessentiality to avoid lethality. While the IS ranked all 784 proteinsidentified in the DEAE separatome, the top six genes that are notconsidered essential, i.e., rfaD, usg, rraA, cutA, nagD, and speA, weredeleted in the initial prototype host cell of this example.

The high priority genes in Table 14 are listed in descending rank order,from greater importance to lesser importance, according to theirimportance score as calculated using Equation 3. The summation includedNaCl fractions 60 mM, 109 mM, 159 mM, 208 mM, 258 mM, 307 mM, 357 mM,406 mM, 456 mM, 505 mM, 1000 mM, b₁=1 for all genes regardless ofessentiality, y_(max)=1000 mM. For h_(i,j), h_(i,total), andh_(j,total), mass spectroscopy data were used to determine the amount ofprotein, which was defined as the number of confident sequencing eventsthat matched to peptides associated with a given protein (h_(i)).MWref=170 kDa (the molecular weight of the largest gene product, mukB);α=1.

The top 50 genes in the IS ranking for purposes of this example areshown in Table 14:

TABLE 14 rfaD usg rraA rpoB rpoC tufA cutA ptsI nagD ycfD speA gldA glnAmetE tgt argG groL prs typA fusA entF hemL ycaO slyD gatZ ilvB glgP nusAmetH gdhA entB prmB rho uvrB infB mukB ilvA metA hslO ppsA recC rnt thiIybiT clpB iscS metL degP rapA purL

This table lists genes ordered by decreasing importance as determinedfrom the importance equation. While only six genes were removed from thewild type E. coli MG1655 parent cell in this experiment to produce theimproved host cell LTSF06, continuing to delete, modify, or inhibit theexpression of genes (while skipping or modifying genes considered to beessential) by moving down the list would continue to reduce the totalnumber of HCPs present in the modified host cell and thus improve E.coli host cells for expression of recombinant peptides, polypeptides,and proteins by improving total column capacity.

Fed-batch culture of LTSF06 demonstrated that growth of this knockoutstrain was not compromised, as similar trajectories of growth unitsversus elapsed fermentation time were obtained for the parent (MG1655)and mutant strain (LTSF06) (FIG. 14).

Lysates of LTSF06 and MG1655 were prepared and loaded to DEAE columns todetermine the total amount of HCPs bound in each case. FIGS. 15, 16, and17 show comparisons of the HCPs bound to DEAE that differ in the amountof NaCl present in the binding buffer, i.e., loading buffer containing 5mM NaCl to minimize non-specific binding. Adding NaCl to the runningbuffer used to equilibrate the column and to the injected lysate iscommon practice to attenuate the column behavior of both HCPs andpotential target protein, so a range of salt concentrations was examinedand included in both low and stringent values. In all cases as shown inFIGS. 15, 16, and 17, there is a favorable difference in the amount ofHCPs bound to the DEAE between the LTSF06 knockout strain compared tocontrol parent E. coli strain MG1655. The reduction in HCPs in knockoutstrain LTSF06 varied between 14% to 17%.

These data demonstrate that the small number of deletions (six)contained in LTSF06, i.e., removal of only 0.119% of the total genome,significantly decreased the total amount of HCPs that would beencountered during target recombinant protein isolation via ion exchangechromatography.

These results demonstrate that the present separatome concept employingthe importance equation provides a novel quantitative and rational meansof identifying and ranking host cell proteins that negatively impactchromatographic separation capacity, and therefore chromatographicselectivity and purity of the final recovered target product. Onceidentified and ranked in this way, such host cell chromatographynuisance proteins can be deleted, modified, or inhibited to produceoptimized host cells for recombinant expression of a broad spectrum oftarget peptides, polypeptides, and proteins, where such cells stillmaintain good (or possibly even improved) fermentation characteristicssuch as growth rates, viability, capacity for expression, etc.

Extrapolating from the results obtained via the six gene deletions inLTSF06, it is reasonable to predict and fully expected that genecombinations containing increased numbers of genes, e.g., 7, 8, 9, 10,and so on similarly up to 50, can be selected from Table 14 fordeletion, modification, and/or inhibition of expression in order toproduce improved E. coli host cells for expression of recombinantpeptides, polypeptides, and proteins. In addition to deletion, etc., ofsequential (contiguous) combinations of high ranking genes listed inTable 14, deletions, etc., of non-sequential and non-contiguouscombinations (which involve “skipping” (omitting) listed genes), andrandom combinations of high ranking genes in this table are alsoencompassed herein. Essential genes that can be modified by the methodsdiscussed below can also be included in any of these gene combinationswhen necessary.

A consideration in designing such combinations involves geneessentiality. Essential genes can be deleted, etc., if the modified hostcells exhibit acceptable viability, growth rates, protein expressionlevels, etc., for the intended application. Alternatively, essentialgenes can be modified, for example, by reducing their expression byreplacing their naturally occurring promoters with weaker promoters,introducing strategic point mutations to replace amino acids involved inresin binding while still maintaining satisfactory levels ofgene/protein activity, or replacing endogenous E. coli genes with genesfrom other organisms that perform the same or similar functions and thatdo not significantly adversely affect chromatographic separationefficiency and separation capacity, or cell growth, viability, andcapacity for expression, rather than deleting them entirely. Suchreplacement genes include heterologs, homologs, analogs, paralogs,orthologs, and xenologs. These strategies facilitate improvements inchromatographic separation efficiency even when interfering host cellproteins include essential genes. In addition, as discussed above in thedefinition of“essential genes”, various feeding strategies can be usedin the present host cells and methods to circumvent potentiallydeleterious effects due to deletion, etc., of essential genes that wouldotherwise adversely impact chromatographic separation efficiency ifpresent.

In addition to sequential and non-sequential, and contiguous andnon-contiguous, deletions of genes listed in Table 14, calculation andidentification of combinations of genes useful in the E. coli host cellsand methods disclosed herein as mathematically described in Example 2are equally applicable to the list of genes disclosed in Table 14 inthis example. That mathematical description and accompanying discussion,including equations 6 and 7, are herein incorporated by reference intheir entirety and applied herein.

The effectiveness of any of the various possible combinations of genestargeted for deletion, etc., selected from Table 14 in improvingchromatographic separation efficiency of target host cell or targetrecombinant peptides, polypeptides, and proteins as described above canbe determined without undue experimentation by the methods disclosedherein.

In summary, the data presented in this example demonstrate that theseparatome concept, including importance equation 3, facilitatesreduction in HCPs encountered during bioprocessing, improving columncapacity and overall chromatographic separation efficiency, withoutadversely impacting host cell growth, viability, or capacity forexpression, and that this can be achieved in a rational, stepwisepredictable way. Results with LTSF06 show that with strategic deletions,significant improvement in column efficiency can be achieved.Identification and ordering of high ranking genes as determined from theimportance equation out of the thousands of genes in the E. coli genomefacilitates maximum improvements in E. coli host cells used forexpression of a wide range of recombinant products without having toengineer individual host cells for specific targets. While otherinvestigations have considered knockout or mutation to improve thepurity of a single recombinant product, the mathematical frameworkdisclosed herein guided minimal changes made to the E. coli genome thatare useful regardless of target recombinant product. These minimal butstrategic changes positively affect the initial chromatographic capturestep, identified as a key bottleneck by polling several biotherapeuticand enzyme manufacturers.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

1. An E. coli host cell for expression of a target host cell or targetrecombinant peptide, polypeptide, or protein, wherein thechromatographic separation efficiency of said target host cell or targetrecombinant peptide, polypeptide, or protein expressed in said E. colihost cell is improved in an amount in the range of from about 5% toabout 50%, said E. coli host cell comprising: a) a reduced genome, b)modified genome, or c) a genome in which expression of genes is reducedor completely inhibited, wherein genes that are deleted, modified, orthe expression of which is reduced or completely inhibited in said hostcell code for peptides, polypeptides, or proteins that impair thechromatographic separation efficiency of said target host cell or targetrecombinant peptide, polypeptide, or protein expressed in said hostcell, wherein said genes are selected from the group consisting of: thegenes listed in Table 9, and combinations thereof; combinations of anyof the genes listed in Tables 8 and 9 taken together; the genes listedin Table 14, and combinations thereof; and combinations of any of thegenes listed in Tables 8, 9, and 14 taken together, wherein deletion,modification, or reduction or complete inhibition of expression of saidgenes improves the chromatographic separation efficiency of said targethost cell or target recombinant peptide, polypeptide, or protein in anamount in the range of from about 5% to about 50% compared tochromatographic separation efficiency of said target host cell or targetrecombinant peptide, polypeptide, or protein in the presence ofpeptides, polypeptides, or proteins coded for by said genes that aredeleted, modified, and/or the expression of which is reduced orcompletely inhibited in said E. coli host cell upon affinity oradsorption, non-affinity column chromatography of said target host cellor target recombinant peptide polypeptide, or protein.
 2. The E. colihost cell of claim 1, which is a strain selected from the groupconsisting of strain K-12, strain B, strain C, and strain W.
 3. The E.coli host cell of claim 2, wherein: said E. coli strain K-12 is selectedfrom the group consisting of W3110, DH10B, DH5alpha, DH1, MG1655, andBW2952; and said E. coli strain B is selected from the group consistingof B REL606, BL21, and BL21-DE3.
 4. The E. coli host cell of claim 1,which is selected from the group consisting of: Alpha-SelectBacteriophage T1-Resistant Gold Efficiency (F− deoR endA1 recA1 relA1gyrA96 hsdR17(rk⁻, mk₊) supE44 thi-1 phoA Δ(lacZYA-argF)U169Φ80lacZΔM15λ−), Alpha-Select Bacteriophage T1-Resistant SilverEfficiency (F− deoR endA1 recA1 relA1 gyrA96 hsdR17(rk⁻, mk₊) supE44thi-1 phoA Δ(lacZYA-argF)U169 Φ80lacZΔM15λ−), Alpha-Select BronzeEfficiency (F− deoR endA1 recA1 relA1 gyrA96 hsdR17(rk−, mk+) supE44thi-1 phoA Δ(lacZYA-argF)U169 Φ80lacZΔM15λ−), Alpha-Select (F− deoRendA1 recA1 relA1 gyrA96 hsdR17(rk−, mk+) supE44 thi-1 phoAΔ(lacZYA-argF)U169 Φ80lacZΔM15λ−), AG1 (endA1 recA1 gyrA96 thi-1 relA1glnV44 hsdR17(r_(K) ⁻ m_(K) ⁺)), AB1157 (thr-1, araC14, leuB6(Am),Δ(gpt-proA)62, lacY1, tsx-33, qsr′-0, glnV44(AS), galK2(Oc), LAM−,Rac-0, hisG4(Oc), rfbC1, mgl-51, rpoS396(Am), rpsL31(strR), kdgK51,xylA5, mtl-1, argE3(Oc), thi-1), B2155 (thrB1004 pro thi strA hsdsSlacZD M15 (F′lacZD M15 lacI^(q) traD36 proA⁺proB⁺) Δ dapA::erm (Erm^(r))pir::RP4 [::kan (Km^(r)) from SM10]), B834(DE3) (F⁻ompT hsdS_(B)(r_(B) ⁻m_(B) ⁻) gal dcm met (DE3)), BIOBlue (recA1 endA1 gyrA96 thi-1hsdR17(rk−, mk+) supE44 relA1 lac [F′ proAB lacI^(q)ZΔM15Tn10(Tet^(r))]), BL21 (E. coli B F− dcm ompT hsdS(r_(B)− m_(B)−) gal[malB⁺]_(K-12)(λ^(S))), BL21(AI) (F⁻ ompT gal dcm lon hsdS_(B) (r_(B) ⁻m_(B) ⁻) araB::T7RNAP-tetA), BL21(DE3) (F⁻ ompT gal dcm lonhsdS_(B)(r_(B) ⁻ m_(B) ⁻) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7nin5])), BL21 (DE3) pLysS (F− ompT hsdSB(rB−, mB−) gal dcm (DE3) pLysS(CamR)), BL21-T1R (F− ompT hsdSB(rB− mB−) gal dcm tonA), BNN93 (F⁻tonA21 thi-1 thr-1 leuB6 lacY1 glnV44 rfbC1 fhuA1 mcrB e14-(mcrA⁻)hsdR(r_(K) ⁻m_(K) ⁺) λ⁻), BNN97 (BNN93 (λgt11)), BW26434(Δ(araD-araB)567, Δ(lacA-lacZ)514(::kan), lacI^(p)-4000(lacI^(q)), λ⁻,rpoS396(Am)?, rph-1, Δ(rhaD-rhaB)568, bsdR514), C600 (F⁻ tonA21 thi-1thr-1 leuB6 lacY1 glnV44 rfbC1 fhuA1λ⁻), CAG597 (F⁻ lacZ(am) pho(am)lyrT[supC(ts)] trp(am) rpsL(Str^(R)) rpoH(am)165 zhg::Tn10 mal(am)),CAG626 (F⁻ lacZ(am) pho(am) lon trp(am) tyrT[supC(ts)] rpsL(Str^(R))mal(am)), CAG629 (F⁻ lacZ(am) pho(am) lon supC(ts) trp(am) rpsLrpoH(am)165 zhg::Tn10 mal(am)), CH3-Blue (F− ΔmcrA Δ(mrr-hsdRMS-mcrBC)Φ80lacZΔM15 ΔlacX74 recA1 endA1 ara Δ139 Δ(ara, leu)7697 galUgalrpsL(Str^(R)) nupG λ−), CSH50 (F⁻ λ⁻ ara Δ(lac-pro) rpsL thifimE::IS1), D1210 (HB101 lacI^(q) lacY⁺), dam-dcm-BacteriophageT1-Resistant (F− dam-13:Tn9(Cam^(R))dcm-6 ara-14 hisG4 leuB6 thi-1 lacY1galK2 galT22 glnV44 hsdR2 xylA5 mtl-1 rpsL136(Str^(R)) rtbD1 tonA31tsx78 mcrA mcrB1), DB3.1 (F− gyrA462 endA1 glnV44 Δ(sr1-recA) mcrB mrrhsdS20(r_(B) ⁻, m_(B) ⁺) ara14 galK2 lacY1 proA2 rpsL20(Sm^(r)) xy15Δleu mtl1), DH1 (endA1 recA1 gyrA96 thi-1 glnV44 relA1 hsdR17(r_(K) ⁻m_(K) ⁺) λ⁻), DH5α Turbo (F′ proA+B+ lacI^(q) Δ lacZ M15/fhuA2Δ(lac-proAB) glnV gal R(zgb-210::Tn10)Tet^(S) endA1 thi-1Δ(hsdS-mcrB)5), DH12S (mcrA Δ(mrr-hsdRMS-mcrBC) φ80d lacZΔM15 ΔlacX74recA1 deoR Δ(ara, leu)7697 araD139 galU galK rpsL F′ [proAB⁺lacI^(q)ZΔM15]), DM1 (F− dam-13::Tn9(Cm^(R)) dcm− mcrB hsdR-M+ gal1 gal2ara− lac− thr− leu− tonR tsxR Su0), E. CLONI® 5ALPHA(fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1endA1 thi-1 hsdR17), E. CLONI® 10G (F− mcrA Δ(mnr-hsdRMS-mcrBC) endA1recA1 Φ80dlacZΔM15 ΔlacX74 araI139 Δ(ara,leu)7697galU galK rpsL nupG λ−tonA (StrR)), E. CLONI® 10GF′ ([F′ pro A+B+ lacI^(q)ZΔM15::T10(Tet^(R))]/mcrA Δ(mrr-hsdRMS-mcrBC) endA1 recA1 Φ80dlacZΔM15 ΔlacX74araD139 Δ(ara, leu)7697 galU galK rpsL nupG λ− tonA (StrR)), E. coli K12ER2738 (F′proA+B+ lacI^(q) Δ(lacZ)M15 zzf::Tn10(Tet^(R))/fhuA2 glnVΔ(lac-proAB) thi-1 Δ(hsdS-mcrB)5), ElectroMax™ DH10B (F⁻mcrAΔ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 endA1araD139Δ(ara,leu)7697 galU galK λ⁻rpsL nupG), ELECTROMAX™ DH5ALPHA-E (F−φ80lacZΔM15 Δ(lacZY A-argF) U169 recA1 endA1 hsdR17 (rk−, mk+) galphoAsupE44λ-thi-1 gyrA96 relA1), ElectroSHOX (F− mcrA Δ(mrr-hsdRMS-mcrBC)Φ80lacZΔM15 ΔlacX74 recA1 endA1 ara Δ139 Δ(ara, leu)7697 galUgalKrpsL(Str^(R)) nupG λ⁻), EP-MAX™10B F′ (mcrA Δ(mrr-hsdRMS-mcrBC)φ80dlacZΔM15 ΔlacX74 deoR recA1 endA1 araD139 Δ(ara, leu)7697 galU galKrpsL nupG λ−/F′[lacI^(q)ZΔM15 Tn10 (Tet^(R))]), ER1793 (F⁻ fhuA2Δ(lacZ)r1 glnV44 e14⁻(McrA⁻) trp-31 his-1 rpsL104 xyl-7 mtl-2 metB1Δ(mcrC-mrr)114::IS10), ER1821 (F⁻ glnV44 e14⁻(McrA⁻) rfbD1? rel4? endA1spoT1? thi-1 Δ(mcrC-mrr))114::IS10), ER2738 (F′proA⁺B⁺ lacI^(q)Δ(lacZ)M15 zzf::Tn10(Tet^(R))/fhuA2 glnV Δ(lac-proAB) thi-1Δ(hsdS-mcrB)5), ER2267 (F′ proA⁺B⁺ lacI^(q) Δ(lacZ)M15 zzf::mini-Tn10(Kan^(R))/Δ(argF-lacZ)U169 glnV44 e14⁻(McrA⁻) rfbD1? recA1 relA1? endA1spoT1! thi-1 Δ(mcrC-mrr)114::IS10), ER2507 (F⁻ ara-14 leuB6 fhuA2Δ(argF-lac)U169 lacY1 glnV44 galK2 rpsL20 xyl-5 mtl-5 Δ(malB)zjc::Tn5(Kan^(R))Δ(mcrC-mrr)_(HB101)), ER2508 (F⁻ ara-14 leuB6 fhuA2Δ(argF-lac)U169 lacY1 lon::miniTn10(Tet^(R)) glnV44 galK2rpsL20(Str^(R)) xyl-5 mtl-5 Δ(malB) zjc::Tn5(Kan^(R))Δ(mcrC-mrr)_(HB101)), ER2738 (F′proA⁺B⁺ lacI^(q) Δ(lacZ)M15zzf::Tn10(Tet^(R))/fhuA2 glnV Δ(lac-proAB) thi-1 Δ(hsdS-mcrB)5), ER2925(ara-14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm-6 hisG4rfbD1 R(zgb210::Tn10)Tet^(S) endA1 rpsL136 dam13::Tn9 xylA-5 mtl-1 thi-1mcrB1 hsdR2), GC5™ (:F− Φ80lacZ Δ M15 Δ (lacZYA-argF)U169 endA1 recA1relA1 gyrA96 hsdR17 (r_(k) ⁻, m_(k) ⁺) phoA supE44 thi-1λ−T1R), GC10 (F−mcrA Δ(mrr-hsdRMSmcrBC) Φ80dlacZ Δ M15 Δ lacX74 endA1 recA1 Δ (ara,leu)7697 araD139 galUgalK nupG rpsL λ−T1R), GENEHOGS® (FmcrAΔ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(araleu)7697 galUgalK rpsL (StrR) endA1 nupG fhuA::IS2 (confers phage T1 resistance)),HB101, HMS174, HMS174(DE3), HI-CONTROL™ BL21(DE3) (F⁻ ompT gal dcmhsdS_(B)(r_(B) ⁻ m_(B) ⁻) (DE3)/Mini-F lacI^(q1)(Gent^(r))), HI-CONTROL™10G (F− mcrA Δ(mrr-hsdRMS-mcrBC) endA1 recA1 Φ80dlacZΔM15 ΔlacX74araD139Δ(ara,leu)7697 galU galK rpsL nupG λ− tonA/Mini-F lacI^(q1) (Gent^(r))),HT96™ NOVABLUE (endA1 hsdR17 (r_(K12) ⁻ m_(K12) ⁺) supE44 thi-1 recA1gyrA96 relA1 lac F′[proA⁺B⁺ lacI^(q)ZΔM15::Tn10] (Tet^(R))), IJ1126,IJ1127, INV110, JM83, JM101 (F′ traD36 proA⁺B⁺ lacI^(q)Δ(lacZ)M15/Δ(lac-proAB) glnV thi), JM103, JM105, JM106, JM107, JM108,JM109 (F′ traD36 proA⁺B⁺ lacI^(q) Δ(lacZ)M15/Δ(lac-proAB) glnV44 e14⁻gyrA96 recA1 relA1 endA1 thi hsdR17), JM109(DE3), JM110, JS5, KS1000 (F′lacI^(q) lac⁺ pro⁺/ara Δ(lac-pro) Δ(tsp)=Δ(prc)::Kan^(R)eda51::Tn10(Tet^(R)) gyrA(Nal^(R)) rpoB thi-1 argE(am)), LE392,Lemo21(DE3) (fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS/pLemo(Cam^(R)) λDE3=λ sBamHIo ΔEcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 Δnin5pLemo=pACYC184-PrhaBAD-lysY), LIBRARY EFFICIENCY® DH5A™ (F−φ80lacZΔM15Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(r_(k) ⁻, m_(k) ⁺) phoA supE44thi-1 gyrA96 relA1λ−), MACH1™ T1R (F− Φ80lacZΔM15 ΔlacX74 hsdR(rK−, mK+)ΔrecA1398 endA1 tonA), MAX EFFICIENCY® DH10B™ (F-mcrAΔ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara,leu)7697 galU galK λ-rpsL nupG/pMON14272/pMON7124), MC1061, MC4100, MDS™42(MGJ655 fhuACDB(del) endA(del)+deletion of 699 additional genes,including all IS elements and cryptic prophages as listed in Posfai etal, (2006) Science (312):1044-1046), MFDpir, NEB Express l^(q1)(MiniFlacI^(q) (Cam^(R))/fhuA2 [lon] ompT gal sulA11R(mcr-73::miniTn10-Tet^(S))2 [dcm] R(zgb-210::Tn10-Tet^(S)) endA1Δ(mcrC-mrr)114::IS10), NEB Express, dam⁻/dcm⁻, NEB 5-alpha (fhuA2Δ(argF-lacZ)U69 phoA glnV44 Φ80Δ (lacZ)M15 gyrA96 recA1 relA1 endA thi-1hsdR17), NEB 10-beta (Δ(ara-leu) 7697 araD139 fhuA ΔlacX74 galK16 galE15e14-φ80dlacZΔM15 recA1 relA1 endA1 nupG rpsL (Str^(R)) rph spoT1Δ(mrr-hsdRMS-mcrBC)), NiCo21(DE3) (can::CBD fhuA2 [lon] ompT gal (λ DE3)[dcm] arnA::CBD slyD::CBD glmS6Ala ΔhsdS λ DE3=λ sBamHIo ΔEcoRI-Bint::(lacI::PlacUV5::T7 gene1) i21 Δnin5), NM522 (F′ proA⁺B⁺ lacI^(q)Δ(lacZ)M15/Δ(lac-proAB) glnV thi-1 Δ(hsdS-mcrB)5), NOVABLUE™ (endA1hsdR17 (r_(K12) ⁻ m_(K12) ⁺) supE44 thi-1 recA1 gyrA96 relA1 lacF′[proA⁺B⁺ lacI^(q)ZΔM15::Tn10](Tet^(R))), NovaF− (F⁻ endA1 hsdR17(r_(K12) ⁻ m_(K12) ⁺) supE44 thi-1 recA1 gyrA96 relA1 lac), NOVAXGF′ZAPPERS™ (mcrA Δ(mcrC mrr) endA1recA1 φ80dlacZΔM15 ΔlacX74araD139Δ(ara-leu)7697 galUgalKrpsLnupGλ⁻tonA F′[lacI^(q)Tn10] (Tet^(R))),OMNIMAX™2T1® (F′ {proAB+ lacIq lacZΔM15 Tn10(TetR) Δ(ccdAB)} mcrAΔ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 Δ(lacZYA-argF), U169 endA1 recA1 supE44thi-1 gyrA96 relA1 tonA panD), ONE SHOT® BL21 STAR™ (DE3) (F−ompT hsdSB(rB−, mB−) galdcmrne131 (DE3)), ONESHOT® TOP10 (F− mcrAΔ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 Δ lacX74 recA1 araD139 Δ(araleu)7697galUgalK rpsL (StrR) endA1 nupG), ORIGAMI™ (Δ(ara-leu) 7697 ΔlacX74 ΔphoAPvuII phoR araD139 ahpC galE galK rpsLF′[lac⁺ lacI^(q) pro](DE3)gor522::Tn10 trxB (Kan^(R), Str^(R), Tet^(R))), ORAGAMI™ 2(Δ(ara-leu) 7697 ΔlacX74 ΔphoA PvuII phoR araD139 ahpC galE galK rpsLF′[lac⁺ lacI^(q) pro] gor522::Tn10 trxB (Str^(R), Tet^(R))),OVEREXPRESS™ C41(DE3) (F− ompT hsdSB (rB− mB−) gal dcm (DE3)),OVEREXPRESS™ C41(DE3)PLYSS (F− ompT hsdSB (rB− mB−) gal dcm (DE3) pLysS(Cm^(R))), OVEREXPRESS™ C43(DE3) (F− ompT hsdSB (rB− mB−) gal dcm(DE3)), OVEREXPRESS™ C43(DE3)PLYSS (F− ompT hsdSB (rB− mB−) gal dcm(DE3) pLysS (Cm^(R))), POP2136/pFOS1 (F⁻ glnV44 hsdR17 endA1 thi-1 aroBmal⁻ cI857 lambdaPR), PR1031 (F⁻ thr:Tn10(Tet^(R)) dnaJ259 leu fhuA2lacZ90(oc) lacY glnV44 thi), ROSETTA™ (F⁻ompT hsdS_(B)(r_(B) ⁻ m_(B) ⁻)gal dcm pRARE (Cam^(R))), ROSETTA™ (DE3)PLYSS (F⁻ompT hsdS_(B)(r_(B) ⁻m_(B) ⁻) gal dcm (DE3) pLysSRARE2 (Cam^(R))), ROSETTA-GAMI™(Δ(ara-leu)7697 ΔlacX74 ΔphoA PvuII phoR araD139 ahpC galE galK rpsLF′[lac⁺ lacI^(q) pro] gor522::Tn10 trxB pRARE2 (Cam^(R), Str^(R),Tet^(R))), ROSETTA-GAMI™ (DE3)PLYSS (Δ(ara-leu)7697 ΔlacX74 ΔphoA PvuIIphoR araD139 ahpC galE galK rpsL (DE3) F′[lac⁺ lacI^(q) pro]gor522::Tn10trxB pLysSRARE2 (Cam^(R), Str^(R), Tet^(R))), RR1, RV308, SCARABXPRESS®T7LAC (MDS™42 multiple-deletion strain (1) with a chromosomal copy ofthe T7 RNA Polymerase gene), SS320 (F′[proAB+lacIqlacZΔM15 Tn10(tet^(r))]hsdR mcrB araD139 Δ(araABC-leu)7679 ΔlacX74 galUgalK rpsLthi), SHUFFLE® (F′ lac pro lacI^(q)/Δ(ara-leu)7697 araD13 fhuA2Δ(lac)X74 Δ(phoA)PvuII phoR ahpC*galE (or U) galK Δλatt::pNEB3-r1-cDsbC(SpecR, lacI^(q)) ΔtrxB rpsL150(StrR) Δgor Δ(malF)3), SHUFFLE® T7 (F′lac, pro, lacI^(q)/Δ(ara-leu)7697 araD139 fhuA2 lacZ::T7 gene1Δ(phoA)PvuII phoR ahpC*galE (or U) galK λatt::pNEB3-r1-cDsbC (Spec^(R),lacI^(q)) ΔtrxB rpsL150(Str^(R)) Δgor Δ(malF)3), SHUFFLE® T7 EXPRESS(huA2 lacZ::T7 gene1 [lon] ompT ahpC gal λatt::pNEB3-r1-cDsbC (Spec^(R),lacI^(q)) ΔtrxB sulA11 R(mcr-73::miniTn10-Tet^(S))2 [dcm]R(zgb-210::Tn10-Tet^(S)) endA1 Δgor Δ(mcrC-mrr)114::IS10), SOLR(e14-(McrA−) Δ(mcrCB-hsdSMR-mrr)171 sbcC recB recJ uvrC umuC::Tn5(Kan^(r)) lac gyrA96 relA1 thi-1 endA1λ^(R) [F′ proAB lacI^(q)ZΔM15]^(C) Su−), SCS110, STBL2™ (F− endA1 gln V44 thi-1 recA1 gyrA96relA1 Δ(lac-proAB) mcrA Δ(mcrBC-hsdRMS-mrr) λ⁻), STBL3™ (F− glnV44recA13 mcrB mrr hsdS20(rB−, mB−) ara-14 galK2 lacY1 proA2 rpsL20 xyl-5leu mtl-1), STBL4™ (endA1 glnV44 thi-1 recA1 gyrA96 relA1 Δ(lac-proAB)mcrA Δ(mcrBC-hsdRMS-mrr) λ⁻ gal F′[proAB⁺ lacI^(q) lacZΔM15 Tn10]),STELLAR™ (F−, endA1, supE44, thi-1, recA1, relA1, gyrA496, phoA, Φ80dlacZΔ M15, Δ (lacZYA-argF) U169, Δ (mrr-hsdRMS-mcrBC), ΔmcrA, λ−), SURE(endA1 glnV44 thi-1 gyrA96 relA1 lac recB recJ sbcC umuC::Tn5 uvrCe14-Δ(mcrCB-hsdSMR-mrr)171 F′[proAB⁺ lacI^(q) lacZΔM15 Tn10]), SURE2(endA1 glnV44 thi-1 gyrA96 relA1 lac recB recJ sbcC umuC::Tn5 uvrCe14-Δ(mcrCB-hsdSMR-mrr) 171 F′[proAB⁺ lacI^(q) lacZΔM15 Tn10 AmyCm^(R)]), T7 Express Crystal (fhuA2 lacZ::T7 gene1 [lon] ompT gal sulA11R(mcr-73::miniTn10-Tet^(S))2 [dcm] R(zgb-210::Tn10-Tet^(S)) endA1 metB1Δ(mcrC-mrr)114::IS10), T7 Express lysY/I^(q) (MiniF lvsYlacI^(q)(Cam^(R))/fhuA2 lacZ::T7 gene1 [lon] ompT gal sulA11R(mcr-73::miniTn10-Tet^(S))2 [dcm] R(zgb-210::Tn10-Tet^(S)) endA1Δ(mcrC-mrr) 114::IS10), T7 Express lysY (MiniF lysY (Cam^(R))/fhuA2lacZ::T7 gene1 [lon] ompT gal sulA11 R(mcr-73::miniTn10-Tet^(S))2 [dcm]R(zgb-210::Tn10-Tet^(S)) endA1 Δ(mcrC-mrr)114::IS10), T7 Express I^(q)(MiniF lacI^(q)(Cam^(R))/fhuA2 lacZ::T7 gene1 [lon] ompT gal sulA11R(mcr-73::miniTn10-Tet^(S))2 [dcm] R(zgb-210::Tn10-Tet^(S)) endA1Δ(mcrC-mrr)114::IS10), T7 Express (fhuA2 lacZ::T7 gene1 [lon] ompT galsulA11 R(mcr-73::miniTn10-Tet^(S))2 [dcm] R(zgb-210::Tn10-Tet^(S)) endA1Δ(mcrC-mrr)114::IS10), TB1 (F⁻ ara Δ(lac-proAB) [Φ80dlac Δ(lacZ)M15]rpsL(Str^(R)) thi hsdR), TG1 (F′ [traD36 proAB⁺ lacI^(q) lacZΔM15]supEthi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5, (r_(K) ⁻m_(K) ⁻)), THUNDERBOLT™ GC10(F− mcrA Δ (mrr-hsdRMSmcrBC) Φ80dlacZ Δ M15 DlacX74 endA1recA1 Δ (ara,leu)7697 araD139 galU galK nupG rpsL l λ-T1R), UT5600 (F⁻ ara-14 leuB6secA6 lacY1 proC14 tsx-67d(ompT-fepC)266 entA403 trpE38 rfbD1 rpsL109xyl-5 mtl-1 thi-1), VEGGIE™ BL21(DE3) (F⁻ompT hsdS_(B)(r_(B) ⁻ m_(B) ⁻)gal dcm(DE3)), W3110 (λ857S7), WM3064, XL1-Blue (endA1 gyrA96(nal^(R))thi-1 recA1 relA1 lac glnV44 F′[::Tn10 proAB⁺ lacI^(q)Δ(lacZ)laM15]hsdR17(r_(K) ⁻ r_(K) ⁺)), XL1-Blue MRF′(Δ(mcrA)183Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F′proAB lacI^(q)ZΔM15 Tn10 (Tet^(r))]), XL2-Blue (endA1 gyrA96(nal^(R))thi-1 recA1 relA1 lac glnV44 F′[::Tn10 proAB⁺ lacI^(q)Δ(lacZ)M15 AmyCm^(R)] hsdR17(r_(K) ⁻ m_(K) ⁺)), XL2-Blue MRF′(endA1 gyrA96(nal^(R))thi-1 recA1 relA1 lac glnV44 e14− Δ(mcrCB-hsdSMR-mrr)171 recB recJ sbcCumuC::Tn5 uvrC F′[::Tn10 proAB⁺ lacI^(q)Δ(lacZ)M15 Amy Cm^(R)]), XL1-Red(F− endA1 gyrA96(nal^(R)) thi-1 relA1 lac glnV44 hsdR17(r_(K) ⁻ m_(K) ⁺)mutS mutT mutD5 Tn10), XL10-Gold (endA1 glnV44 recA1 thi-1 gyrA96 relA1lac Hte Δ(mcrA)183 Δ(mcrCB-hsdSMIR-mrr)173 tet^(R) F′[proABlacI^(q)ZΔM15 Tn10(Tet^(R) Amy Cm^(R))]), and XL10-Gold KanR (endA1glnV44 recA1 thi-1 gyrA96 relA1 lac Hte Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 tet^(R) F′[proAB lacI^(q)ZΔM15 Tn10(Tet^(R) Amy Tn5(Kan^(R))]). 5.The E. coli host cell of claim 1, wherein the number of saidcombinations of said genes either for Table 8 alone, Table 9 alone,Tables 8 and 9 together, Table 14, or for Tables 8, 9, and 14 togetheris determined by combination Equation 6: $\begin{matrix}\frac{n!}{{r!}{\left( {n - r} \right)!}} & {{Equation}\mspace{14mu} 6}\end{matrix}$ wherein n is the set of genes out of which selectionoccurs, and r is the number of genes selected for deletion,modification, and/or inhibition.
 6. The E. coli host cell of claim 1,wherein said combinations of said genes are selected from the groupconsisting of: ΔhldDΔusgΔrraA; ΔhldDΔusgΔrraAΔcutA;ΔhldDΔusgΔrraAΔcutAΔnagD; ΔhldDΔusgΔrraAΔcutAΔnagDΔspeA;ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldA;ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnA;ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetE;ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgt;ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargG;ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypA;ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentF;ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaO;ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyD;ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgatZ;ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgatZΔilvB;ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgatZΔilvBΔ glgP;ΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgatZΔilvBΔglgPΔnusA; andΔhldDΔusgΔrraAΔcutAΔnagDΔspeAΔgldAΔglnAΔmetEΔtgtΔargGΔtypAΔentFΔycaOΔslyDΔgatZΔilvBΔglgPΔnusAΔmetH, wherein in any of the foregoing gene combinationscomprising multiple genes, one or more of these genes is omitted as longas the resulting E. coli host cells exhibit growth rates, viability, orcapacity for expression of target molecules in the range of from about60% to about 100%, or more, of that of the same E. coli host cell inwhich said gene combinations are not deleted, not modified, or theexpression of which is not reduced or completely inhibited, and as longas chromatographic separation capacity is improved in an amount in therange of from about 5% to about 50%, or more, compared to that of thesame E. coli host cell in which said gene combinations are not deleted,not modified, or the expression of which is not reduced or completelyinhibited, depending on the number and particular combination of genesdeleted, modified, and/or inhibited, or wherein any of the foregoinggene combinations also further comprises deletion, modification, orinhibition or reduction of expression of one or more essential genesselected from among rpoB, rpoC, tufa, ycfD, groL, prs, fusA, hemL, slyD,infB, mukB, and rnt as long as the resulting E. coli host cells exhibitgrowth rates, viability, or capacity for expression of target moleculesin the range of from about 60% to about 100%, or more, of that of thesame E. coli host cell in which said gene combinations are not deleted,not modified, or the expression of which is not reduced or completelyinhibited, and as long as chromatographic separation capacity isimproved in an amount in the range of from about 5% to about 50%, ormore, compared to that of the same E. coli host cell in which said genecombinations are not deleted, not modified, or the expression of whichis not reduced or completely inhibited, depending on the number andparticular combination of genes deleted, modified, or inhibited. 7.(canceled)
 8. (canceled)
 9. The E. coli host cell of claim 1, whereinsaid chromatographic separation efficiency is independent of elutionconditions under which said target host cell or target recombinantpeptide, polypeptide, or protein emerges from an affinity or adsorption,non-affinity chromatography column as an enriched fraction. 10.(canceled)
 11. (canceled)
 12. (canceled)
 13. (canceled)
 14. The E. colihost cell of claim 1, wherein said target host cell or targetrecombinant peptide, polypeptide, or protein is present in a lysate ofsaid E. coli host cell, or is secreted by said E. coli host cell. 15.The E. coli host cell of claim 1, wherein said target host cell peptide,polypeptide, or protein or target recombinant peptide, polypeptide, orprotein is an endogenous peptide, polypeptide, or protein.
 16. The E.coli host cell of claim 1, wherein said target recombinant peptide,polypeptide, or protein is a heterologous peptide, polypeptide, orprotein.
 17. The E. coli host cell of claim 16, wherein saidheterologous peptide, polypeptide, or protein is selected from the groupconsisting of an enzyme and a therapeutic peptide, polypeptide, orprotein.
 18. The E. coli host cell of claim 17, wherein said enzyme isselected from the group consisting of a nuclease, a ligase, apolymerase, an RNA- or DNA-modifying enzyme, a carbohydrate-modifyingenzyme, an isomerase, a proteolytic enzyme, and a lipolytic enzyme, andsaid therapeutic peptide, polypeptide, or protein is selected from thegroup consisting of antibody, an antibody fragment, a vaccine, anenzyme, a growth factor, a blood clotting factor, a hormone, a nervefactor, an interferon, an interleukin, tissue plasminogen activator, andinsulin.
 19. A method of preparing a pharmaceutical or veterinarycomposition comprising a therapeutic peptide, polypeptide, or protein,comprising the steps of: i) expressing said therapeutic peptide,polypeptide, or protein in said E. coli host cell of claim 1; ii) in thecase where said therapeutic peptide, polypeptide, or protein is notsecreted from said E. coli host cell, preparing a lysate of said hostcell containing said therapeutic peptide, polypeptide, or protein,producing an initial therapeutic peptide-, polypeptide-, orprotein-containing mixture; or iii) in the case where said therapeuticpeptide, polypeptide, or protein is secreted from said E. coli hostcell, harvesting culture medium in which said host cell is grown,containing said therapeutic peptide, polypeptide, or protein, therebyobtaining an initial therapeutic peptide-, polypeptide-, orprotein-containing mixture; iv) chromatographing said initialtherapeutic peptide-, polypeptide-, or protein-containing mixture ofstep ii) or step iii) via affinity or adsorption-based, non-affinitychromatography and collecting elution fractions, thereby obtaining oneor more fractions containing an enriched amount of said therapeuticpeptide, polypeptide, or protein relative to other peptides,polypeptides, or proteins in said fraction compared to the amount ofsaid therapeutic peptide, polypeptide, or protein relative to otherpeptides, polypeptides, or proteins in said initial protein mixture; v)optionally, further chromatographing an enriched fraction of step iv) toobtain said therapeutic peptide, polypeptide, or protein in a desireddegree of purity; and vi) recovering said therapeutic peptide,polypeptide, or protein.
 20. The method of claim 19, further comprisingformulating said therapeutic peptide, polypeptide, or protein with apharmaceutically or veterinarily acceptable carrier, diluent, orexcipient to produce a pharmaceutical or veterinary composition,respectively.
 21. The method of claim 19, wherein said adsorption-based,non-affinity chromatography is ion exchange chromatography. 22.(canceled)
 23. A method of preparing a pharmaceutical or veterinarycomposition comprising a therapeutic peptide, polypeptide, or protein,comprising the steps of: i) expressing said therapeutic peptide,polypeptide, or protein in said E. coli host cell of claim 6; ii) in thecase where said therapeutic peptide, polypeptide, or protein is notsecreted from said E. coli host cell, preparing a lysate of said hostcell containing said therapeutic peptide, polypeptide, or protein,producing an initial therapeutic peptide-, polypeptide-, orprotein-containing mixture; or iii) in the case where said therapeuticpeptide, polypeptide, or protein is secreted from said E. coli hostcell, harvesting culture medium in which said host cell is grown,containing said therapeutic peptide, polypeptide, or protein, therebyobtaining an initial therapeutic peptide-, polypeptide-, orprotein-containing mixture; iv) chromatographing said initialtherapeutic peptide-, polypeptide-, or protein-containing mixture ofstep ii) or step iii) via affinity or adsorption-based, non-affinitychromatography and collecting elution fractions, thereby obtaining oneor more fractions containing an enriched amount of said therapeuticpeptide, polypeptide, or protein relative to other peptides,polypeptides, or proteins in said fraction compared to the amount ofsaid therapeutic peptide, polypeptide, or protein relative to otherpeptides, polypeptides, or proteins in said initial protein mixture; v)optionally, further chromatographing an enriched fraction of step iv) toobtain said therapeutic peptide, polypeptide, or protein in a desireddegree of purity; and vi) recovering said therapeutic peptide,polypeptide, or protein.
 24. The method of claim 23, further comprisingformulating said therapeutic peptide, polypeptide, or protein with apharmaceutically or veterinarily acceptable carrier, diluent, orexcipient to produce a pharmaceutical or veterinary composition,respectively.
 25. The method of claim 23, wherein said adsorption-based,non-affinity chromatography is ion exchange chromatography.
 26. A methodof purifying an enzyme, comprising the steps of: i) expressing saidenzyme in said E. coli host cell of claim 1; ii) in the case where saidenzyme is not secreted from said E. coli host cell, preparing a lysateof said host cell containing said enzyme, producing an initialenzyme-containing mixture; or iii) in the case where said enzyme issecreted from said E. coli host cell, harvesting culture medium in whichsaid host cell is grown, containing said enzyme, thereby obtaining aninitial enzyme-containing mixture; iv) chromatographing said initialenzyme-containing mixture of step ii) or step iii) via affinity oradsorption-based, non-affinity chromatography and collecting elutionfractions, thereby obtaining one or more fractions containing anenriched amount of said enzyme relative to other peptides, polypeptides,or proteins in said fraction compared to the amount of said enzymerelative to other peptides, polypeptides, or proteins in said initialprotein mixture; v) optionally, further chromatographing an enrichedfraction of step iv) to obtain said enzyme in a desired degree ofpurity; and vi) recovering purified enzyme.
 27. A method of purifying anenzyme, comprising the steps of: i) expressing said enzyme in said E.coli host cell of claim 6; ii) in the case where said enzyme is notsecreted from said E. coli host cell, preparing a lysate of said hostcell containing said enzyme, producing an initial enzyme-containingmixture; or iii) in the case where said enzyme is secreted from said E.coli host cell, harvesting culture medium in which said host cell isgrown, containing said enzyme, thereby obtaining an initialenzyme-containing mixture; iv) chromatographing said initialenzyme-containing mixture of step ii) or step iii) via affinity oradsorption-based, non-affinity chromatography and collecting elutionfractions, thereby obtaining one or more fractions containing anenriched amount of said enzyme relative to other peptides, polypeptides,or proteins in said fraction compared to the amount of said enzymerelative to other peptides, polypeptides, or proteins in said initialprotein mixture; v) optionally, further chromatographing an enrichedfraction of step iv) to obtain said enzyme in a desired degree ofpurity; and vi) recovering purified enzyme.